Effective detection of dopamine (DA) and uric acid (UA) has significant diagnostic values, while the analytical methods with high convenience, distinguishability and sensitivity still need to be developed. A highly sensitive and selective electrochemical sensor based on a polyaniline (PANI) nanocomposite embedding Au and Cu2O hybrid (PANI/Cu2O-Aux) with adjustable proportions of Cu2O and Au was developed for simultaneous detection of dopamine (DA) and uric acid (UA) in the presence of high concentrations of ascorbic acid (AA) and other interferents. The Cu2O-Aux architecture improved the electrocatalytic activity by inducing a modifiable synergistic effect between Au and Cu2O. Differential pulse voltammetry (DPV) responses of DA and UA on modified electrodes of PANI/Cu2O-Aux showed remarkable enhancement compared to the single-component Au or Cu2O-decorated PANI. The optimal ratio of Cu2O: Au was 1:1, which gave the highest redox signals for DA and UA. The DPV response ranges of simultaneous detection were 0.01–200 μM for DA and 0.1–1000 μM for UA, with detection limits (S/N = 3) of 0.0076 μM and 0.035 μM, respectively. The sensor also exhibited excellent anti-interference and stability, and was successfully applied to measure DA and UA in human urine samples with high accuracy. This research demonstrates the tunability of catalytic properties in PANI/Cu2O-Aux nanocomposites by the integration of binary nanocatalysts, providing insights into both structural and practical aspects.