In this work, Pt/reduced graphene oxide nanocomposite (Pt/RGO) was prepared by in situ chemical reduction. The Pt/RGO modified glassy carbon electrode exhibited excellent catalytic activity towards the oxidation of dopamine (DA) and uric acid (UA) in the presence of ascorbic acid (AA). Differential pulse voltammetry (DPV) was used to simultaneous determination of DA and UA with high sensitivity. •Pt/reduced graphene oxide nanocomposite was prepared by in situ chemical reduction.•Pt nanoparticles were well-dispersed on reduced graphene oxide sheets.•The as-prepared nanocomposite displayed high electrocatalytic activity.•It was used to simultaneous determination of dopamine and uric acid in the presence of ascorbic acid with high sensitivity. In this paper, a Pt/reduced graphene oxide (Pt/RGO) modified glassy carbon electrode was prepared for the detection of dopamine (DA) and uric acid (UA) in the presence of high concentration of ascorbic acid (AA). The electrochemical behavior of the Pt/RGO modified electrode was characterized by cyclic voltammetry and differential pulse voltammetry, which showed good performance toward individual detection of DA and UA and even their simultaneous detection in the presence of 1.0mM AA. Evidently, the electro-oxidation peak currents displayed linear relationship with the associated DA and UA concentrations in the range of 10.0-170.0 μM and 10.0-130.0 μM, respectively, with the detection limits of 0.25 μM for DA and 0.45 μM for UA at three folds of the signal-to-noise ratio. The good performance of the Pt/RGO modified electrode provided a promising alternative in routine sensing applications.