The attraction is housed inside a replica of 30 Rockefeller, where The Tonight Show is taped. The corridors of the actual NBC Studios are also replicated in the queue. Divided in two parts, the queue is composed of one in a lower level and the second in the upper. As guests enter the lobby in the lower level, they're given a colored ticket at the receptionist desk, one of the six colors of the NBC peacock logo. The lower level features displays of various pieces of The Tonight Show's 62-year history from each host. Artifacts include suits of the past hosts, desk props, admittance tickets from each host's tenure and an NBC video camera used for the show's taping in the golden age, all adjacent to a display of Jimmy Fallon as the current host. Guests may continue to the upper level once the lobby's lighting changes to the color of the NBC ticket they were given.
In the upper level of the queue, guests are entertained with a live performance from The Ragtime Gals barbershop quartet onstage, with screens showing Jimmy's monologues, show sketches, History of Rap and Lip Sync Battle. The queue also has interactive desks throughout the waiting area where guests can send personalized Tonight Show Thank You Notes, play retro-style Fallon video games and have a meet-and-greet with Hashtag the Panda.
Guests are called in to enter Studio 6B once the lighting in the upper level waiting area changes to the color of the NBC ticket they were given at the receptionist desk. After guests are ushered into the doors that lead to Studio 6B, The Tonight Show Starring Jimmy Fallon begins in the screens. The show begins with its intro and music played by The Roots, with Steve Higgins announcing the guests for this episode: you (the audience) "and featuring the Legendary Roots Crew". The shot cuts to Questlove Yelling "Universal Studios!" followed by Higgins introducing Jimmy Fallon. As he emerges from the curtains, studio applause lights flash, indicating the guests to do so as the show intro proceeds.
After the intro ends, Fallon begins the show with a quick monologue about the sights the audience will get to see in the race through New York City and requests the help of Roots vocalist Tariq Trotter to rap about the safety precautions of the ride, instead of reciting them because "that's boring". After the applause lights flash again for The Roots, Sara and her stepdad, Gary, make an appearance as the only people in the studio audience, who later leave the studio for the ride's gift shop. After they leave, Fallon ushers guests to step inside Studio 6B to begin the race, as he returns backstage through the curtains he emerged from to board his vehicle, "The Tonight Rider" (a nod to Knight Rider, which aired on NBC).
The race through New York begins with Jimmy Fallon emerging once again from backstage through the stage curtains, this time driving "The Tonight Rider", challenging the audience to the race. The Roots can be seen to provide the music as Steve Higgins counts down the start of the race. The race begins through Studio 6B's backstage corridors, racing through NBC personnel, passing by Tight Pants and Hashtag the Panda. After almost crashing into Hashtag, Jimmy's vehicle skids through the corridor and crashes through the wall along with the audience, landing at West 50th Street between the NBC Studios marquee and Radio City Music Hall. From there, the race immediately continues through Times Square, after Jimmy crashes into a taxi driven by Travis Bickle (Fallon).
The race continues under the Hard Rock Cafe marquee and heads towards the New York City Subway's Times Square–42nd Street station, where the audience encounters The Ragtime Gals singing a melody before turning to the subway tracks and almost crashing with an oncoming train (operated by Fallon). The audience gets to escape by reversing and turning into a tunnel under construction and later exiting to the first checkpoint, where Sara is crossing the street with her stepdad, Gary, who's carrying a basket full of the Tonight Show Golden Retriever puppies. Jimmy Fallon's wife, Nancy Juvonen makes a cameo appearance with their daughters, Winnie and Frances Fallon.
The race now takes the skies as Jimmy's vehicle converts into a hovercraft, soaring through the city, throwing pizza to the audience and t-shirts out of a t-shirt cannon. The race goes past the Flatiron Building before diving into the East River for the second leg of the race, where a shark appears and scares Fallon and the audience (a reference to Jaws). After resurfacing, the race reaches the Statue of Liberty for the third checkpoint.
As fireworks explode around Lady Liberty, the race heads towards the Empire State Building where a giant Hashtag the Panda, trying to flick off planes, grabs the audience and shoots them straight to the moon. There, Jimmy remarks The Roots as "the first band on the moon", who are all dressed up in spacesuits. He then, asks James Poyser to play his thank you note-writing music, to write a thank you note addressed to the moon. The race reaches the Space Train, a roller coaster launcher on the moon, which is under construction, shooting both Jimmy and the audience back towards Earth. Coming in too hot, Jimmy decides to open up a hyperdrive in the form of a tube that leads directly to 30 Rock and back inside Studio 6B, where the audience wins the race after Jimmy Fallon crashes his "Tonight Rider" just before crossing the finish line.
The Tonight Show's real-life stage manager, Niclana Tolmasoff, makes a cameo appearance at the end of the ride as well as the show's real-life writers, Mike DiCenzo and Gavin Purcell, who appear as stage crew members.
The Tonight Shop Edit
After the ride concludes, guests exit downstairs to The Tonight Shop, with merchandise from both the ride and Fallon's Tonight Show tenure.