A three-dimensional g-C3N4/MWNTs/GO hybrid modified electrode was constructed as an electrochemical sensor for the simultaneous determination of ascorbic acid (AA), dopamine (DA) and uric acid (UA). Due to the high conductivity of MWCNTs and the strong synergy between g-C3N4 and GO, the combination of the three effectively improved the electrocatalytic activity of the modified electrode for the oxidation of AA, DA, and UA, and solved the problems such as overlapping anodic peaks. The electrochemical performance of the as-constructed sensor was investigated and optimized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The linear response range of AA, DA, and UA in the optimal condition was 0.2–7.5 mM, 2–100 μM, and 4–200 μM, respectively. The detection limits (S/N = 3) of AA, DA, and UA were 96, 0.22, and 1.36 μM, respectively. The recoveries of AA, DA and UA in serum samples from three groups were 92.82–106.50%, and the relative standard deviations were less than 2%. The results show that the as-constructed g-C3N4/MWNTs/GO modified electrode has the advantages of simplicity, high sensitivity and good selectivity, and can simultaneously determine AA, DA, and UA. Display omitted •A three-dimensional g-C3N4/MWNTs/GO hybrid electrode was constructed as an electrochemical sensor.•g-C3N4/MWNTs/GO hybrid sensor can simultaneously determine DA, AA, and UA.•The recoveries of DA, AA and UA in serum samples from three groups were 92.82–106.50%.•g-C3N4/MWNTs/GO hybrid sensor has the advantages of simplicity, high sensitivity and good selectivity.