Abstract: In order to obtain the optimal culture conditions and volatile components of Trametes versicolor mycelium, a single factor experimental method was used to study the effects of different carbon sources, nitrogen sources, pH values, medium volume, culture temperature, and culture time on the production of volatile components in T. versicolor mycelium. The culture conditions were further optimized by response surface methodology and the volatile components of T. versicolor mycelium were analyzed by gas chromatography-mass spectrometry. The results showed that the volume of culture medium, culture time, and culture temperature had a significant impact on the volatile components of T. versicolor mycelium. The optimal culture conditions were sucrose as the carbon source, no nitrogen source added, 120 mL of culture medium volume, 27.5 ℃ of culture temperature, and 27 days of culture time. Under these conditions, the error between the validation test results with the theoretical prediction is 0.315%. A total of 6 volatile components were detected from the mycelium of T. versicolor, including 3 alcohols, 1 aromatics, and 2 sesquiterpenes. Among them, 1-epibicyclosesquiphellandrene and (E)-calamenene were the main components, with relative contents of 29.96% and 29.17%, respectively. This provides a basis for the further development and application of T. versicolor mycelium in microbial fragrances.