Abstract Photo‐oxidase nanozymes are emerging enzyme‐mimicking materials that produce reactive oxygen species (ROS) upon light illumination and subsequently catalyze the oxidation of the substrate. Carbon dots are promising photo‐oxidase nanozymes due to their biocompatibility and straightforward synthesis. Carbon dot‐based photo‐oxidase nanozymes become active for ROS generation under UV or blue light illumination. In this work, sulfur and nitrogen doped carbon dots (S,N‐CDs) were synthesized by solvent‐free, microwave assisted technique. We demonstrated that sulfur, nitrogen doping of carbon dots (band gap of 2.11 eV) has enabled photo‐oxidation of 3,3,5,5′‐tetramethylbenzidine (TMB) with extended visible light (up to 525 nm) excitation at pH 4. The photo‐oxidase activities by S,N‐CDs produce Michaelis–Menten constant (K m ) of 1.18 mM and the maximum initial velocity (V max ) as 4.66×10 −8 Ms −1 , under 525 nm illumination. Furthermore, visible light illumination can also induce bactericidal activities with growth inhibition of Escherichia coli ( E. coli ). These results demonstrate that S,N‐CDs can increase intracellular ROS in the presence of LED light illumination.