Abstract: Hydroquinone (HQ) was used to catalyze Methanobactin (Mb) to reduce chlorauric acid to prepare Gold Nanoparticles (AuNPs). Mb@AuNPs was modified onto the surface of a bare gold electrode via electrodeposition to prepare the Mb@AuNPs/Au electrode. Subsequently, differential pulse voltammetry was conducted for the simultaneous determination of nitrite and ascorbic acid concentrations as well as condition optimization. For the electrode assembly, electrodeposition was performed with a 0.20 mol/L and pH6.5 PBS at a 100 mV/s scan rate for 40 cycles. In the linear range of 2~5600 μmol/L and 1~6000 μmol/L, the oxidation peak current of both nitrite and ascorbic acid detected by Mb@AuNPs/Au showed a good linear relationship with the concentration (R²>0.9928), and the limit of detection (LOD) was 0.31 and 0.57 μmol/L, respectively. The recovery rates of standard addition for the actual samples were 92.59%~109.26%, 90.01%~103.51%. The method has good reproducibility and stability and can be used for simultaneous determination of nitrite and ascorbic acid in fruits and vegetables.