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 58.1 kDa was observed on SDS‐PAGE of the purified enzyme. Km values were calculated as 0.25, 0.40, and 0.83 mM for 4‐MC, DHPPA, and L‐DOPA, respectively. The highest Vmax 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 Ki 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. Practical applications A small number of wild mushrooms can be used as food, and some of them are traditionally used in the treatment of certain diseases. L. laccata (wild edible mushroom) is first time reported as a PPO source. The results obtained in this study may be useful for understanding the behavior of mushroom catecholases. In addition, LacPPO may be used in the production of compounds which can be synthesized in the presence of organic solvents such as food additives and pharmaceutical drugs in need of natural compounds since the enzyme has the ability to catalyze the reaction in the environment of the organic solvent.