Precise profiling of biocatalytic transformations is of critical importance for diagnosis and treatment. Herein, taking advantage of tyrosinase-triggered polymerization of tyrosine, a fluorescence universal strategy based on gold nanoclusters (AuNCs) for monitoring H₂O₂-generated biocatalytic reactions was designed. In this approach, melanin-like polymer facilely formed by virtue of oxidation of tyrosine monomers, which employed as efficient quencher for fluorescence of AuNCs, contributing to a photoinduced electron-transfer mechanism. Such fluorescence responds can be obviously inhibited by H₂O₂, enabling the sensitive sensing of H₂O₂in a cost-effective and time-consuming way. Harnessing well-defined polymer system and H₂O₂-generated biocatalytic processes enabled sensitive detection of cholesterol and acetylcholine (substrates) with detection limit of 100 nmol L-1 and 500 nmol L-1 in low background and convenient manner. Meanwhile, the present bioassay exhibits excellent specificity to metabolite on account of the specificity of enzyme. This versatile and modular platform incorporates highly efficient catalytic reaction of enzyme into AuNCs@melanin-like polymer system, enhancing its potential uptake into clinical diagnostics for accurate profiling the metabolites that participated in H₂O₂-generation reactions.