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Hydrogen sulfide (H₂S), an endogenous gasotransmitter, is produced in mammalian systems and is closely associated with pathological and physiological functions. Nevertheless, the complete conversion of H₂S is still unpredictable owing to the limited number of sensors for accurate and quantitative detection of H₂S in biological samples. In this study, we constructed a disposable electrochemical sensor based on PtNi alloy nanoparticles (PtNi NPs) for sensitive and specific in situ monitoring of H₂S released by human breast cancer cells. PtNi alloy NPs with an average size of 5.6 nm were prepared by a simple hydrothermal approach. The conversion of different forms of sulfides (e.g., H₂S, HS⁻, and S²⁻) under various physiological conditions hindered the direct detection of H₂S in live cells. PtNi NPs catalyze the electrochemical oxidation of H₂S in a neutral phosphate buffer (PB, pH 7.0). The PtNi-based sensing platform demonstrated a linear detection range of 0.013–1031 µM and the limit of detection was 0.004 µM (S/N = 3). Moreover, the PtNi sensor exhibited a sensitivity of 0.323 μA μM⁻¹ cm⁻². In addition, the stability, repeatability, reproducibility, and anti-interference ability of the PtNi sensor exhibited satisfactory results. The PtNi sensor was able to successfully quantify H₂S in pond water, urine, and saliva samples. Finally, the biocompatible PtNi electrode was effectively employed for the real-time quantification of H₂S released from breast cancer cells and mouse fibroblasts.