Abstract: Objective:To establish a determination method of the content of methanobactin (Mb) obtained by fermentation of M. trichosporium OB3b during fermentation process and fermentation ripening stage. Methods:The effects of addition of CuSO₄, HDTMA, phosphate buffer and complexation time on the content of Mb were investigated. Based on the determination of Mb content in freeze-dried powder after fermentation by chrome azurol S method, in order to control the Mb fermentation process, the Mb content of every 24 h in the first 4 days during fermentation was determined by high performance liquid chromatography. Results:The content of CuSO₄, HDTMA and phosphate buffer was 1.2, 4.0, 2.0 mL and the complexation time was 15 min, the content of Mb in the purified 1 L fermentation broth was 26.91 μmolL, and the minimum detection limit was 0.042 μmol/L.Combining with high performance liquid chromatography, the Mb content in the fermentation broth was determined to be 1.008, 1.336, 3.125, 7.500 μmol/250 mL at 24, 48, 72 and 96 h, respectively. Conclusion:The method established was stable and had good reproducibility, which could be used for the determination of Mb content in different stages of fermentation.