Abstract l-Tyrosine (Tyr), playing roles as both a reducing reagent and a protecting ligand, has been first employed for synthesizing fluorescent gold nanoclusters (AuNCs@Tyr) via a novel one-pot strategy. The as-prepared AuNCs@Tyr exhibited a fluorescence emission at 470nm with a quantum yield of approximately 2.5%. Subsequently, the AuNCs@Tyr described here was applied for detections of tyrosinase (TR) activity, which was based on the mechanism of aggregations of AuNCs@Tyr occurring on the active sites of TR since TR was introduced, thus leading to the fluorescence quenching of AuNCs@Tyr. Accordingly, TR was analyzed in a linear range of 0.5–200umL−1 with a detection limit of 0.08umL−1 at a signal-to-noise ratio of 3. Significantly, TR has been considered as a critical marker for melanoma owing to its specifically expressing in melanoma cells. Therefore, this analytical method towards investigating TR activity may broaden avenues for meaningfully clinical applications.