A carbon paste electrode (CPE) modified with gold nanoparticles was used as a selective electrochemical sensor for the determination of tryptophan (Trp), uric acid (UA) and ascorbic acid (AA). Cyclic voltammetric, chronoamperometric, and differential pulse voltammetric methods were used to investigate the electrocatalytic oxidation of Trp in aqueous solution. The Au nanoparticles modified carbon paste electrode (AuNPs/CPE) showed very efficient electrocatalytic activity for anodic oxidation of Trp, UA, and AA. The linear calibration range for Trp in the presence of 100 μM AA and 50 μM UA was 6–200 μM, with a detection limit of 0.65 μM (S/N = 3). A linear relationship was found for UA in the range of 6–180 μM containing 100 μM AA and 25 μM Trp, with a detection limit of 0.71 μM. The linear calibration range for AA in the presence of 50 μM UA and 50 μM Trp was 20–250 μM, with a detection limit of 2.05 μM. The diffusion coefficient (D) for the oxidation of Trp at the modified electrode was calculated as 2.82 × 10⁻⁵ cm² s⁻¹. The modified electrode showed good sensitivity and selectivity, and was employed for the determination of Trp, UA, and AA in biological and pharmaceutical samples.