Hydrogen peroxide (H₂O₂), as a biomarker in a series of physiological processes, plays a crucial role in investigating the pathogenesis of some diseases. The highly sensitive and selective detection of H₂O₂ release from living cells has aroused tremendous research interests. Herein, a non-enzymatic electrochemical H₂O₂ biosensor was constructed based on AgPt bimetallic nanoparticle-structured MoS₂ nanohybrid (AgPt/MoS₂) as an electrocatalyst. The AgPt/MoS₂ nanohybrid was successfully synthesized by a facile wet-chemical method. Compared with AgPt nanoparticles, the AgPt/MoS₂ nanohybrid exhibited significant enhancement in catalytic performance towards H₂O₂. The main factor is that MoS₂ nanosheets as a supporting material promote AgPt nanoparticles to expose more active sites. The electrochemical response of the AgPt/MoS₂ hybrid-based biosensor to H₂O₂ was linear in the range of 20 μM-4 mM, and the detection limit was calculated as 1.0 μM. To prove practicality, the biosensor was further applied to detect H₂O₂ release from living SMMC-7721 cells. These results indicate the potential applications of the developed biosensor in monitoring physiological processes.