A novel electrochemical sensor based on dual-template molecularly imprinted polymer (MIP) with nanoporous gold leaf (NPGL) was established for the simultaneous determination of dopamine (DA) and uric acid (UA). NPGL acts as an enlarged loading platform to enhance sensing capacity, and the MIP layer was synthesized in situ in the presence of monomer and dual templates (DA and UA) to provide specific recognition. Under the optimal conditions, the sensor shows a good linear range of 2.0180 μM for DA at a working potential of 0.15 V (vs. Ag/AgCl) and 5.0160 μM for UA at 0.35 V (vs. Ag/AgCl), with the respective detection limit of 0.3 μM and 0.4 μM (S/N = 3). Good selectivity of the sensor to its dual templates was confirmed as the sensing signals are significantly different between templates and interfering species. The responses maintained higher than 96% of the initial values after 30-day storage, and the day-to-day relative standard deviation is less than 3.0%. Real sample simultaneous determination of DA and UA was conducted with bovine serum, and the results were in good agreement with those from high-performance liquid chromatography. It can be concluded that this work offers a reliable, facile, fast, and cost-effective method of simultaneous quantification of two or more chem-/bio-molecules. Graphical abstract.