Single-atom catalysts (SACs) attract widespread attention in heterogeneous catalysis due to their maximum atomic utilization efficiency and unique physical and chemical properties. However, their applications in chemical sensing keep huge potential but remain unclear. Herein, a Ni-N₄-C SAC was synthesized for the trace detection of dopamine (DA) and uric acid (UA). The Ni-N₄-C SAC exhibited superior sensing performance compared to the Ni clusters. The detection range for DA and UA were 0.05–75 µM and 5–90 µM with detection limits of 0.027 and 0.82 µM, respectively. Density functional theory (DFT) computations indicate that Ni-N₄-C has a lower reaction barrier during electrochemical process, indicating that the atomic Ni sites possess higher intrinsic activity than Ni clusters. Moreover, DA and UA show strong potential dependency on the Ni-N₄-C catalyst, indicating its applicability for their concurrent detection. This work extends the application of SACs in chemical sensing.