Abnormal hydrogen peroxide (H2O2) levels in the cellular environment are closely related to cell dysfunction and serious diseases. Thus, selective and sensitive H2O2 detections are urgently needed for clinical diagnosis and therapy. Herein, via surface defect engineering, an oxygen‐tolerant electrocatalyst based on tin oxide for selective H2O2 reduction and detection with exceptional stability and activity is designed and developed. When introduced at an appropriate level (≈5.3%), surface oxygen vacancies help lower the charge transfer resistance for enhancing the H2O2 reduction reaction, while maintain the weak oxygen (O2) adsorption, which enables a constant H2O2 reduction (sensing) response in the electrolyte at variable oxygen levels. Moreover, the tin oxide‐based assay system exhibits outstanding stability over a wide pH range of 4–9, as well as selectivity in the presence of interferent endogenous and exogenous electroactive species, which is suitable for trace H2O2 monitoring secreted from NB4 cells, a model cancer cell. The oxygen vacancy‐mediated tin oxide achieves the highest stability as well as high selectivity compared to reported electrochemical probes for specific H2O2 detection in biological environments, with the potential for biological and biomedical applications.