For the study of cell biology, real-time information on cell physiological processes will be more accurate and closer to the in vivo condition in a three-dimensional (3D) culture system. Although most reported 3D cell culture scaffolds can better mimic the in vivo dynamic microenvironment, the real-time analysis technique is deficient or lacking. Herein, a stretchable and conductive 3D scaffold is developed to construct an electrochemical biosensor for real-time monitoring of cell release in 3D culture under stimulation of drug stimulant and mechanical force. In our design, the polyurethane sponge (PU) dipped with conductive carbon ink (CC/PU) was used as a conductive scaffold, and gold nanoparticles (nano-Au) were electrodeposited on the CC/PU (nano-Au CC/PU) to improve the electrochemical sensing performance. The prepared nano-Au CC/PU scaffold exhibits a good electrocatalytic ability to H2O2 with a linear range from 20 nM to 43 μM. Due to the great biocompatibility, HeLa cells can be cultured directly on the nano-Au CC/PU and the in situ and real-time tracking of H2O2 secretion from cells was achieved. The results demonstrate that both the drug stimulant and mechanical force can rapidly activate the release of reactive oxygen species. This study indicates that the stretchable 3D sensing scaffold has good potential for cell biology research in an in vivo-like microenvironment and can be extensively used in the fields of tissue engineering, drug screening, and pathological research.