A practical sensor based on a glassy carbon electrode (GCE) modified with Ag nanoparticles (AgNPs), graphene oxide (GO) and poly(L-arginine) (P(Arg)) was prepared to determine ascorbic acid (AA), dopamine (DA), uric acid (UA) and L-tryptophan (L-Trp) levels simultaneously via differential pulse voltammetry. The new GCE/AgNPs/P(Arg)-GO electrode exhibited high electrocatalytic activity for AA, DA, UA and L-Trp. Scanning electron microscopy (SEM) was utilized to characterize the surface morphology of the composite electrode. Electrochemical characterizations of the bare and composite electrodes were carried out via cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). According to differential pulse voltammetry (DPV) results, there were linear relationships between the peak currents and the concentrations in the ranges of 4.0-2400.0 mu mol L-1 for AA, 0.05-50.0 mu mol L-1 for DA, 0.5-150.0 mu mol L-1 for UA and 1.0-150 mu mol L-1 for L-TRP, with the detection limits (3 s/m) of 0.984, 0.01, 0.142 and 0.122 mu mol L-1 for AA, DA, UA and L-TRP, respectively. The modified electrode could eliminate the interference effects of Na+, K+, L-lysine, glucose, L-cysteine, urea and citric acid. Furthermore, the sensor was successfully applied for the detection of these substances in the human urine samples, and it showed a very high recovery percentage. The comparison of this method with official method also affirmed the accuracy of the data obtained by GCE/AgNPs/P(Arg)-GO.