Gold nanoclusters (AuNCs) have shown great potential in mimicking the redox properties of nanozymes. However, limited fundamental knowledge and a lack of an effective means have hindered the enhancement of their catalysis efficiency. In this study, we report a simple protocol for the synthesis of D-trytophan methyl ester protected AuNCs and exhibit their peroxidase-like activity in a chromogenic reaction coupling with 3,3′,5,5′-tetramethylbenzidine-H2O2. Interestingly, the addition of tetracycline resulted in a remarkable acceleration of the redox reaction rate with AuNCs as the nanozymes due to the increased levels of reactive oxygen species. The UV–vis adsorption intensity of oxidized 3,3′,5,5′-tetramethylbenzidine presented a good linear relationship with the concentration of tetracycline in the range of 1.5–30.0 µM and the limit of detection of the method was 0.2 µM. The proposed assay was further applied in the analysis of serum tetracycline. This study enriches our basic understanding of the enhancement of the catalytic efficiency of AuNCs-based nanozymes.