Herein, a novel tunable electrocatalytic nanobiointerface for the construction of a high‐sensitivity and high‐selectivity biofuel‐cell (BFC)‐based self‐powered biosensor for the detection of transcription factor protein p53 is reported, in which bilirubin oxidase (BOD)/DNA supramolecular modified graphene/platinum nanoparticles hybrid nanosheet (GPNHN) works as a new enhanced material of biocathode to control the attachment of target, and thus tune the electron‐transfer process of oxygen reduction for transducing signaling magnification. It is found that in the presence of p53, the strong interaction between the wild‐type p53 and its consensus DNA sequence on the electrode surface can block the electron transfer from the BOD to the electrode, thus providing a good opportunity for reducing the electrocatalytic activity of oxygen reduction in the biocathode. This in combination with the glucose oxidation at the carbon nanotube/Meldola’s blue/glucose dehydrogenase bioanode can result in a current/or power decrease of BFC in the presence of wild‐type p53. The specially designed BFC‐based self‐powered p53 sensor shows a wide linear range from 1 pM to 1 μM with a detection limit of 1 pM for analyzing wild‐type p53. Most importantly, our BFC‐based self‐powered sensors can detect the concentrations of wild‐type p53 in normal and cancer cell lysates without any extensive sample pretreatment/separation or specialized instruments. The present BFC‐based self‐powered sensor can provide a simple, economical, sensitive, and rapid way for analyzing p53 protein in normal and cancer cells at clinical level, which shows great potential for creating the treatment modalities that capitalize on the concentration variation of the wild‐type p53. Circuit breaker: A new tunable electrocatalytic nanobiointerface for the construction of a high‐sensitivity and ‐selectivity biofuel‐cell‐based, self‐powered biosensor for transcription factor protein p53 detection is demonstrated (see figure). The sensor exhibits significant advantages over the conventional assays in terms of cost efficiency, stability, and speed, especially without the need of any large and expensive instruments in laboratories.