Given the significance of uric acid and creatinine in clinical diagnostic, disease prevention and treatment, a multifunctional electrochemical sensor was proposed for sensitive detection of uric acid and creatinine. The sensitive detection of uric acid was realized based on the unique electrochemical oxidation of nanoporous gold (NPG) towards uric acid, showing good linearity from 10 μM to 750 μM with a satisfactory sensitivity of 222.91 μA mM cm and a limit of detection (LOD) of 0.06 μM. Based on the Jaffé reaction between creatinine and picric acid, the sensitive detection of creatinine was indirectly achieved in a range from 10 to 2000 μM by determining the consumption of picric acid in the Jaffé reaction with a detection sensitivity of 195.05 μA mM cm and a LOD of 10 μM. For human urine detection using the proposed electrochemical sensor, the uric acid detection results were comparable to that of high-performance liquid chromatography (HPLC), with a deviation rate of less than 10.28% and the recoveries of uric acid spiked in urine samples were 89~118%. Compared with HPLC results, the deviation rate of creatinine detection in urine samples was less than 4.17% and the recoveries of creatinine spiked in urine samples ranged from 92.50% to 117.40%. The multifunctional electrochemical sensor exhibited many advantages in practical applications, including short detection time, high stability, simple operation, strong anti-interference ability, cost-effectiveness, and easy fabrication, which provided a promising alternative for urine analysis in clinical diagnosis.