Monooxygenase domain of cytochrome P450BM-3 from Bacillus megaterium was evolved by error-prone PCR. Three mutants (D168N, A225V, K440N; E435D; I39V) with higher hydroxylating activity than the parent type P450BM-3((A74G, F87V, L188Q)) were obtained, coupled with a sensitive screening method of absorption of hydroxylating indole to indigo at 630nm. The catalytic activities of three mutants were 6.6(hml001), 9.6(hml002), 5.3 (hml003) fold higher than that of the parent type P450BM-3 respectively. The kinetic analysis revealed that the mutant enzymes exhibit a higher substrate binding ability and catalytic efficiency than the parent enzyme. DNA sequence indicated that hml001 and hml003 cover one amino acid substitution (I39V and E435D, respectively), hml002 contains three amino acid substitutions (D168N, A225V, K440N).