Recently, in situ detection of hydrogen peroxide (H2O2) generated from live cells have caused tremendous attention, because it is of great significance in the control of multiple biological processes. Herein, Fe-N-C single-atom nanozymes (Fe-N-C SAzymes) with intrinsic peroxidase-like activity were successfully prepared via high-temperature calcination using FeCl2, glucose, and dicyandiamide as precursors. The Fe-N-C SAzymes with FeNx as active sites were similar to natural metalloproteases, which can specifically enhance the peroxidase-like activity rather than oxidase-like activity. Accordingly, owing to the excellent catalytic efficiency of the Fe-N-C SAzymes, colorimetric biosensing of H2O2 in vitro was performed via a typical 3,3',5,5'-tetramethylbenzidine induced an allochroic reaction, demonstrating the satisfactory specificity and sensitivity. With regard to the practical application, in situ detection of H2O2 generated from the Hela cells by the Fe-N-C SAzymes was also performed, which can expand the applications of the newborn SAzymes.