Electropolymerization of Im on the GCE, the PIm modified electrode was denoted as PIm/GCE. Subsequently, the PIm/GCE was washed with doubly distilled water, and then transferred to 0.1M PBS (pH 4.0) for electrochemical oxidation at +1.8V for 250s. The obtained electrode was denoted as PImox/GCE (Fig. A). Then, the deposition of GNPs on PImox/GCE was carried out. The obtained electrode was described as GNPs/PImox/GCE (Fig. B). Display omitted ► The electropolymerization of imidazole (Im) on GCE was first reported. ► The PIm film can be overoxidized to form the overoxidized polyimidazole (PImox). ► PImox allows dispersing of Au and generates additional electrocatalytic sites. ► The overlapping voltammetric response of AA, DA, UA and Trp is well-resolved. A novel electrode was developed through electrodepositing gold nanoparticles (GNPs) on overoxidized-polyimidazole (PImox) film modified glassy carbon electrode (GCE). The combination of GNPs and the PImox film endowed the GNPs/PImox/GCE with good biological compatibility, high selectivity and sensitivity and excellent electrochemical catalytic activities towards ascorbic acid (AA), dopamine (DA), uric acid (UA) and tryptophan (Trp). In the fourfold co-existence system, the peak separations between AA–DA, DA–UA and UA–Trp were large up to 186, 165 and 285mV, respectively. The calibration curves for AA, DA and UA were obtained in the range of 210.0–1010.0μM, 5.0–268.0μM and 6.0–486.0μM with detection limits (S/N=3) of 2.0μM, 0.08μM and 0.5μM, respectively. Two linear calibrations for Trp were obtained over ranges of 3.0–34.0μM and 84.0–464.0μM with detection limit (S/N=3) of 0.7μM. In addition, the modified electrode was applied to detect AA, DA, UA and Trp in samples using standard addition method with satisfactory results.