The ongoing search by the characters for the unknown third man in the spy ring is a take-off of the real-life years-long search for an unknown "Fifth Man" in the so-called Cambridge Five, a British spy ring which began operating during WWII.
The writers had decided not to bring back Bough, English's sidekick from the 2003 film, as played by Ben Miller and decided to replace him with new character Agent Tucker. Miller did film a cameo, to link the two films together, but was cut.
The shown chemical formula of Timoxeline Barbebutenol, the fictional mind control drug pictured during the film, is actually the one of a barbiturate, any of a class of organic compounds used in medicine as sedatives, as hypnotics, or as an adjunct in anesthesia. Amobarbital and Pentobarbital are real-life drugs that share that formula.
The jacket and tie combination worn by Rowan Atkinson during the scenes set in Mozambique are almost identical to those he wore in the unofficial James Bond movie Never Say Never Again (1983) almost thirty years earlier, in which he also featured as an inept secret agent.
"Royce", the V16 Rolls Royce Phantom Coupe was developed specifically for the Johnny English Reborn film at the behest of Rowan Atkinson, a renowned petrol-head. He went to BMW and requested they put the V16 engine originally slated for the production V12 Phantom Limo into their 100EX coupe prototype
The character Patch Quartermain, played by Tim McInnerny, is confined to a wheelchair due to an accident. McInnerny played the character Max in Notting Hill whose wife, Bella (played by Gina McKee) was confined to a wheelchair due to an accident. McInnerny, Notting Hill writer Richard Curtis and Rowan Atkinson all worked together on the 1980's sitcom Blackadder.
In this film, Johnny is undergoing martial arts training with monks in Tibet. 3 years after the film's release, Rowan Atkinson returned as his most famous iconic character Mr. Bean in a Snickers advert. In that advert, Mr. Bean (Atkinson) is a hopeless Kung Fu warrior.
Daniel Kaluuya had previously appeared in the 2009 "Doctor Who" special "Planet of the Dead" as Barclay. Rowan Atkinson had played The Doctor in the 1999 Comic Relief parody "Doctor Who: The Curse of Fatal Death".