Biological desulfurization is a process in which sulfur compounds are removed from gas and oil using microorganisms. It is a simple process that has mild operating conditions, high desulfurization efficiency, low energy consumption and less environmental pollution. However, there is still a lack of simple and efficient analytical methods for quantitatively analyzing the sulfur compounds in the biological desulfurization process. In order to solve this problem, the analytical method for the simultaneous determination of sulfite, thiosulfate and sulfide in biological desulfurization solutions by pre-column fluorescence derivation using high performance liquid chromatography (HPLC) was developed. The standard curves of sulfur species in this analytical method had good linear relationships with correlation coefficients of 0.999 5, 0.999 7, and 0.999 7 for sulfite, thiosulfate and sulfide, respectively. The detection limits of these sulfur compounds were 0.000 6, 0.000 7 and 0.001 1 μmol/L; the range of recovery rates were 98.17 to 101.9%, 100.9 to 102.6%, and 101.1 to 104.2%; which had good repeatability and stability. The analytical method was simple, efficient and accurate, and could be used to simultaneously determine the sulfur compounds in different biological desulfurization systems.