Hydrothermal synthesis of nanocomposites is of significant importance, as it affords facile, biocompatible, nontoxic, and economic fabrication. Herein, we report a hitherto unexplored cytocompatible and reusable biomimetic electrochemical sensor based on pyridyl porphyrin functionalized nitrogen doped graphene nanosheets. The porphyrin functionalized nitrogen doped graphene nanosheets (PFNGS) were prepared by a low temperature hydrothermal method via non-covalent strategies with a minimal impact on their physicochemical properties. Owing to their exceptional attributes like operational ease, low cost, portability, and sensitivity, the as-synthesized PFNGS, formed by π-π interactions, were employed for sensing nitric oxide (NO), which is a key regulator of diverse biological processes. Compared to porphyrin and nitrogen doped graphene nanosheets alone, PFNGS exhibited exceptional sensitivity (3.6191 μA μM-1) and remarkable electrocatalytic properties (0.61 V). This clearly outperforms the previously reported modified electrode materials for the electrochemical detection of NO. Cyclic voltammetry (CV) data also suggested that the PFNGS modified electrode possessed an increased reactive surface area, which results in an increase in the number of reactive sites and low charge transfer resistance. These results also demonstrated that the PFNGS modified electrode showed high stability and reproducibility, the limit of detection (LOD) (S/N = 3) of which was estimated to be 1 nM. Our PFNGS were found to be highly biocompatible and could also detect NO released from macrophage cells. This blend of biocompatibility, electrode stability, electrocatalytic activity along with enhanced sensitivity and selectivity makes PFNGS a powerful and reliable nanomaterial for various biomedical applications in complex biological systems.