Simultaneous electrochemical detection of dopamine (DA) and hydroquinone (HQ) is clinically significant but challenging because overlapping redox signals obscure detection on conventional sensing materials. Single-atom catalysts (SACs) offer promising adsorption and electron-transfer capabilities; however, limited synthetic control, poor cycling durability, and inevitable signal interference in complex multi-analyte matrices hamper their application. Herein, we report a nickel single-atom catalyst embedded in nitrogen-doped carbon (Ni SA/NC) with a unique electronic structure that enables simultaneous detection of DA and HQ. Benefiting from the unique Ni–N₄ moieties, the oxidation peaks for DA and HQ are well resolved, enabling simultaneous quantification. Notably, the Ni SA/NC achieves ultra-low detection limits (0.0018 μM for DA and 0.0017 μM for HQ), wide segmented linear response windows, and high anti-interference selectivity toward typical coexisting contaminants. Additionally, the Ni SA/NC exhibits outstanding measurement reproducibility, as evidenced by ultra-low relative standard deviations, alongside favorable storage stability. DFT calculations reveal that the Ni–N₄ configuration modulates the Ni d-band center to afford moderate *OH adsorption strength, thereby lowering the electro-oxidation barriers. Overall, this work establishes a rational design paradigm for SAC-based electrochemical sensing materials, offering potential for practical implementations in early disease diagnosis.