In recent decades, studies have focused on inorganic nanozymes to overcome the intrinsic drawbacks of bioenzymes due to the demands of improving the reaction conditions and lack of robustness to harsh environmental factors. Many biochemical reactions catalyzed by enzymes require light activation. Light-activated nanozymes have distinct advantages, including being regulated by light stimuli, activating the molecular oxygen to produce reactive oxygen species (ROS) without interfering supplementary oxidants, and often showing a synergistic effect to catalyze some challenging reactions. Only a few studies have been done on this connection. Therefore, it is still a big challenge to develop a nanozyme regulated by light activation. Herein, we uncovered the light-activated oxidase mimicking activity of a conducting polymer polyaniline nanofibers (PANI-NFs). PANI-NFs exhibit intrinsic light-activated brilliant oxidase-like activity, can catalyze the colorless tetramethyl benzidine (TMB) to produce a blue product TMBox, and have a distinct Km = 0.087 mM and a high Vmax = 2.32 μM min–1 value, measured by using Hanes–Woolf kinetics. We also report the light-activated oxidase activity of some other renowned carbocatalysts graphene oxide and graphitic carbon nitride and compare them with PANI-NFs. This type of property shown by the conductive polymer is amazing. The density functional theory is used to verify the stability and the mode of adsorption of the PANI NFs-TMB composite, which corroborates the experimental results. Furthermore, the current nanozyme demonstrated a significant ability to kill both Gram-negative and Gram-positive bacteria as well as effectively destroy biofilms under physiological conditions. We believe that this work provides the motivation to create a link between optoelectronics and biological activity in the near future.