Over the past few decades, metal nanoparticles have been actively investigated as enzyme mimetic nanomaterials. However, the catalytic activity of gold nanocluster (AuNCs)-based nanozymes is relatively low. It is still a great challenge to improve the enzyme-mimic catalytic property of AuNCs, and to explore the roles of the charges on the surface of the nanozymes and reactive oxygen species in the catalytic reaction systems. This study describes a simple synthesis of AuNCs capped with papain (P@AuNCs). The as-prepared P@AuNCs exhibited an efficient peroxidase-mimic ability via the catalytic oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide. Interestingly, the negatively charged dopamine was able to trigger the aggregration of the positively charged P@AuNCs and reactive oxygen species generated in the oxidation process, resulting in a remarkable catalytic activity promotion of P@AuNCs. Based on this principle, a protocol for the highly selective and sensitive monitoring of dopamine has been constructed with the colour change from pale blue to deep blue. The ultraviolet-visible absorbance of P@AuNCs-TMB at the wavelength of 650 nm showed a good linear relationship with the dopamine concentration ranging from 2.0 μM to 25.0 μM (R2 = 0.990). The limit of detection was 0.8 μM. Furthermore, dopamine was monitored in a drug metabolic process following the abdominal injection in rats using the proposed colorimetric assay. It offers an easy approach for the fabrication of AuNCs-based nanozymes with an improved catalytic activity, and provides a great potential application in the measuring of real serum drugs.