Existing electrochemical biosensing platforms, using traditional rigid and unstretchable electrodes, cannot monitor the biological signaling molecules released by cells in a mechanically deformed state in real time. Here, a stretchable and flexible electrochemical sensor was developed based on nickel metal-organic framework composite/Au nanoparticle-coated carbon nanotubes (Ni-MOF composite/AuNPs/CNTs) for sensitive detection of dopamine (DA) released by C6 living cells in real time. A Ni-MOF composite was obtained by introducing Ni, NiO, and a carbon frame onto the surface of two-dimensional (2D) Ni-MOF nanosheets using an efficient one-step calcination method. The hybrid of Ni-MOF composite/AuNPs/CNTs that deposited on the poly(dimethylsiloxane) (PDMS) film endowed the sensor with excellent electrochemical performance with a wide linear range of 50 nM to 15 μM and a high sensitivity of 1250 mA/(cm M) and also provided the sensor with desirable stability against mechanical deformation. Furthermore, the stretchable electrode also displayed good cellular compatibility while C6 living cells can be cultured and proliferated on it with strong adhesion. Then, the DA released by C6 living cells with chemical induction in both natural and stretched states was monitored using our stretchable and flexible electrochemical sensor in real time. This indicates that our new design of flexible Ni-MOF composite/AuNPs/CNTs/PDMS (NACP) film electrodes provides more opportunities for the detection of chemical signals released from cells and soft living organisms even under mechanically deformed states.