In this study, catecholase was purified from Laccaria laccata by affinity chromatography and the enzyme activity was investigated in organic media. Among the tested substrates, the highest catecholase activity was observed in 4-MC, DHPPA, and L-DOPA. A single band around 58.1kDa was observed on SDS-PAGE of the purified enzyme. Km values were calculated as 0.25, 0.40, and 0.83mM for 4-MC, DHPPA, and L-DOPA, respectively. The highest V-max value was calculated as 2500 U/mg protein for 4-MC. The inhibitors used in this study showed mixed type inhibition kinetics for all tested substrates. Ascorbic acid was found to be most effective inhibitor with low K-i value. Also, it was observed that LacPPO is more stable at optimum pH and temperature. In addition, LacPPO was found to be an effective biocatalyst in organic medium. According to the results obtained, the purified enzyme may be useful for some industrial and/or clinical purposes.