Objective: To establish a method for determination of the content of arbutin in microbe conversion solution by Thin-Layer chromatography. Methods: The microbe conversion solution was filtrated and condensed. The developing solvent was a mixture of ethyl acetate, acetic acid and water (3.1:1:1, v/v/v). The detection wavelength was set at 570 nm. Results: The calibration curve was Linear in the range of 2 μg~6 μg with r value of 0.9976. Conclusions: The method was quick, simple and accurate for the determination of content of arbutin.