Professor Mystery is the son of the local Seattle couple, Denise and Bernie, who was obsessed with containing a black hole that they discovered, but they disappeared for some unknown reason, leaving Mystery to fend for himself in Seattle. Because of this, it had led him to a life of evil and being a mad scientist in general. This also led the O.W.C.A. to assign agent Peter the Panda as his nemesis. However, despite their rivalry, it turns out that both Mystery and Peter had a fall-out because of Mystery's lack of communication, even refusing to tell Peter about his tragic upbringing, as he puts it, "Mystery is my allure".
Upon learning that the local Danville mad scientist Heinz Doofenshmirtz has been filling his role as Peter's temporary nemesis, Mystery felt angry and blamed Doofenshmirtz for 'stealing' his nemesis. He then decided to get rid of him, though it was more likely in order to get Peter's attention so that he can go back to being Peter's nemesis. First up, Mystery head over to Danville infiltrated into Doofenshmirtz Evil Incorporated, where he spotted Doofenshmirtz blogging on his computer. He blindfolded him, and then ate a sandwich that Doofenshmirtz was saving for lunch before heading back to his lair in Seattle. After placing Doof in a capsule, he revealed himself, assuming that Doofenshmirtz knows about him thanks to Peter, but Doofenshmirtz stated that he doesn't since Peter doesn't talk. As Mystery transported Doofenshmirtz into a different room, Doofenshmirtz learned about Mystery's lack of communication with Peter and tried to convince him into talking to Peter to set things right. However, sticking to his 'mystery' comment, Professor Mystery refused and intended to finish Doofenshmirtz off with an unnamed -inator he created. However, catching wind of the situation, Peter arrived in battle Mystery, and Doofenshmirtz forced him to explain what the -inator does. Having no choice, Mystery finally confessed that it's an Unexistinator, designed to erase anything from existence, which has left Doofenshmirtz horrified. Fortunately, Doofenshmirtz's nemesis, Perry the Platypus, arrived in time to save Doofenshmirtz by pointing the inator up into the ceiling before it fired. The ray went through the roof and bounced off the Space Needle, and headed over to Danville, where it hits a capsule, which actually contained the same black hole that Mystery's parents were trying to contain. Because of this, the capsule was erased from existence, along with the black hole, thus the world was safe again.
Despite his plan being foiled, Mystery is still relieved that he finally got Peter's attention. They both then head over to a coffee shop, where Mystery explains his backstory to Peter, and finally sees how much the communication is worth it. Around the same time, Bernie and Denise have arrived, recognizing Mystery as their long-lost son. Seeing this his parents are alive, Mystery embraces them in a hug, happy to see them again, and that his backstory is finally resolved. Declaring that their work is done, Doofenshmirtz and Perry (who were both watching this) happily head back to Danville in peace. It is unknown what happened to Mystery afterward.