Owing to the unique coordination environment and high atom utilization efficiency, single atom catalysts have been considered as an ideal artificial enzyme to mimic natural enzymes. Herein, single-atom Fe nanozyme anchored on N-doped Ti₃C₂T_x (Fe SA/N-Ti₃C₂T_x) with asymmetrically coordinated Fe-N₁C₂ configuration is synthesized by vacancy capture and heteroatom doping strategy, which exhibits excellent peroxidase-like activity. Based on the results of peroxidase catalytic kinetics and X-ray adsorption fine spectroscopy, the Fe-N₁C₂ active sites in Fe SA/N-Ti₃C₂T_x are responsible for the excellent performance. Furthermore, the developed Fe SA/N-Ti₃C₂T_x can be employed to quantitative detection of melatonin (MT), which shows a wide linear detection range (0.01–100 μM) and an excellent detection limit (7.3 nM) in buffer, 0.01–100 μM and 7.8 nM in serum samples. Our work proves that MXene-based single atoms can be promising nanozyme in the field of bioassays.