3`-Hydroxygenistein can be obtained from the biotransformation of genistein by the engineered Pichia pastoris X-33 strain, which harbors a fusion gene composed of CYP57B3 from Aspergillus oryzae and a cytochrome P450 oxidoreductase gene (sCPR) from Saccharomyces cerevisiae. P. pastoris X-33 mutants with higher 3`-hydroxygenistein production were selected using a periodic hydrogen peroxide-shocking strategy. One mutant (P2-D14-5) produced 23.0 mg/l of 3`-hydroxygenistein, representing 1.87-fold more than that produced by the recombinant X-33. When using a 5 L fermenter, the P2-D14-5 mutant produced 20.3 mg/l of 3`-hydroxygenistein, indicating a high potential for industrial-scale 3`-hydroxygenistein production.