Hydrogen sulfide (H2S), as the third gas transporter in biological systems, plays a key role in the regulation of biological cells. Real-time detection of local H2S concentration in vivo is an important and challenging task. Herein, we explored a novel and facile strategy to develop a flexible and transparent H2S sensor based on gold nanowire (AuNW) and carbon nanotube (CNT) films embedded in poly(dimethylsiloxane) (PDMS) (AuNWs/CNTs/PDMS). Taking the advantage of the sandwich-like nanostructured network of AuNWs/CNTs, the prepared electrochemical sensing platform exhibited desirable electrocatalytic activity toward H2S oxidation with a wide linear range (5 nM to 24.9 μM) and a low dete ction limit (3 nM). Furthermore, thanks to the good biocompatibility and flexibility of the sensor, HeLa cells can be cultured directly on the electrode, allowing real-time monitoring of H2S released from cells under a stretched state. This work provides a versatile strategy for the construction of stretchable electrochemical sensors, which has potential applications in the study of H2S-related signal mechanotransduction and pathological processes.