Tyrosinase is the key enzyme for melanogenesis with both monophenolase activity and diphenolase activity, which catalyzes the hydroxylation of tyrosine to L-DOPA and the further oxidation of DOPA, respectively. A continuous assay method was developed to directly monitor the real monophenolase activity using synchronous fluorescence. Complexation with borate to quench the native fluorescence of DOPA could selectively quantified the tyrosine in the binary mixture of tyrosine and DOPA under the wavelength difference Δλ = 67 nm for synchronous fluorescence. The limit of detection (LOD) for tyrosine were estimated to be 0.49 μM. Borate was used as a trapping agent for DOPA to abolish diphenolase activity, while hydroxylamine was used as a reducing agent to restore the catalytic cycle. The time course for consumption of tyrosine was established by monitoring the tyrosine fluorescence intensity at discrete intervals of 30 s. Calibration curve between monophenolase activity and tyrosinase concentration with range from 0.1830 U·mL-1 to 1.7034 U·mL-1, and LOD of 0.0721 U·mL-1. Using the proposed method, the Km and υmax for monophenolase was determined with values of 20.73 μM and 1.10 μM·min-1, respectively. Zinc ion was demonstrated to inhibit the monophenolase activity by competitive inhibition manner with IC50 of 14.36 μM. The assay method displayed a powerful application in kinetics and inhibitor screening for monophenolase.