The method of inducement-in situ spectra analysis was developed and used to directly detect and characterize microsome P450 and its inducement in Phanerochaete chrysosporium (PC) by n-hexane, benzoic acid, naphthalene and pentachlorophenol at molecular level. The results demonstrate that the content of P450 after inducement by n-hexane, benzoic acid, naphthalene reached a high level of 7.5, 5.6 and 3.1 times of that without inducement, respectively. The inducement of P450 in fungi by pentachlorophenol has not been reported before this study. The substrate binding spectra of benzoic acid with P450 from PC grown in both low nitrogen medium and PDB medium, and NADPH-dependent degradation of benzoic acid in PC microsome were detected, which provide a direct evidence of the function of P450 to catabolize benzoic acid. However, the inhibition was not observed during the degradation of benzoic acid by PC with P450 inhibitor aminobenzotriazole, which indicates that other catabolic pathway may also exist in PC.