An activated glassy carbon electrode(A-GCE) was prepared by using simple cyclic voltammetry(CV) scanning clean glassy carbon electrode(GCE) in 1.0 mol?L-1 H2SO4 solution.In 0.1 mol?L-1 pH 7.0 phosphate buffer solution(PBS),A-GCE not only improved the oxidations of ascorbic acid(AA),dopamine(DA),uric acid(UA) and nitrite(NO-2),but also resolved the weak and overlapped voltammetric responses of AA,DA,UA and NO-2 into four sensitive oxidation peaks.The peak potential differences(ΔEp,a) of AA-DA,DA-UA and UA-NO-2 were 0.16 V,0.15 V and 0.45 V,respectively,indicating that A-GCE has a good electro-catalytic response towards AA，DA，UA and NO-2.Thus,the A-GCE could be used for the simultaneous determination of AA,DA,UA and NO-2 in their mixture.Under the optimized experimental conditions,the linear dependences of DPV current responses for AA,DA,UA and NO-2 were observed in the concentration ranges of 5-2 100，2-140，1-700,10-1 050 μmol?L-1,with correlation coefficients of 0.998 9，0.996 8，0.997 4 and 0.997 8,respectively.The detection limits for AA,DA,UA and NO-2 were 0.19，0.45，0.28,0.72 μmol?L-1,respectively.The A-GCE was applied in the simultaneous determination of AA,DA,UA and NO-2 in serum samples with satisfactory results.