ABSTRACTTyrosinase is a binuclear copper‐containing metalloprotein that leads the fast and regio‐selective o‐hydroxylation of monophenols to o‐diphenols. However, the subsequent second oxidation to produce o‐quinones, i.e., melanin precursors, from the o‐diphenols has restricted its use to the production of functional o‐diphenol derivatives. Herein, we present a combined strategy for the effective inhibition of melanin formation in tyrosinase reaction, which allows the use of tyrosinase as a monophenol monooxygenase. The o‐diphenolic products were protected from being oxidized in the tyrosinase reaction by borate ions and L‐ascorbic acid (LAA). Borate‐o‐diphenol complexes were favorable formed at high pH and consequentially protected the o‐diphenolic products from the catecholase activity of tyrosinase. LAA not only directly reduced the byproduct, o‐quinones, into o‐diphenols but also assisted the completion of the tyrosinase reaction cycle by removing a hydroxyl group attached to the copper metal cluster at the active site of the met‐form tyrosinase. The regio‐selective o‐hydroxylation of 7,4'‐dihydroxyisoflavone (daidzein) to produce 7,3',4'‐trihydroxyisoflavone (3'‐ODI) was successfully carried out by whole E. coli cell biotransformation with heterologously expressed tyrosinase from Bacillus megaterium. The yield of this o‐hydroxylation of 5 mM daidzein in one‐pot 400 mL reaction was ca. 100% in 90 min and the productivity was 16.3 mg 3'‐ODI · L−1 · h−1 · DCW mg−1, which is considerably higher than that of other monooxygenases. The method effectively abolished melanin synthesis, so that the o‐diphenolic product remained stable without enzyme inactivation. Other monophenolic phytochemicals such as resveratrol and genistein could be subjected to the same strategy. After 1 h, 1 mM of genistein and resveratrol were both converted to orobol and piceatannol, respectively, with ca. 95% conversion yield. These results support the strong potential of tyrosinase as a monooxygenase for regio‐selective o‐hydroxylation of various monophenolic compounds. Biotechnol. Bioeng. 2016;113: 735–743. © 2015 Wiley Periodicals, Inc.