An effective reversed-phase high performance liquid chromatographic method has been developed to separate industrial 3,3'-diamino diphenyl sulfone and its related substances. Separation was achieved on a reversed-phase C18 column with methanol-water (40/60, v/v) as the mobile phase and detection was operated by UV absorption at a wavelength of 250 nm. One unknown impurity, 3,4'-diamino diphenyl sulfone, was identified by the combination of liquid chromatography-mass spectrometry and relationship between RP-HPLC retention and hydrophobicity of the compounds studied. Partial related impurities, 4,4'-diamino diphenyl sulfone, 3,4'-diamino diphenyl sulfone and 3-amino-3'-nitro diphenyl sulfone were determined under the proposed chromatographic condition with relative standard deviations lower than 4.4 %. On the basis of good separation and reliable determination of related substances in 3,3'-diamino diphenyl sulfone, the chromatographic fingerprint of industrial 3,3'-diamino diphenyl sulfone was established, by which quality assessment of 3,3'-diamino diphenyl sulfone could be facilitated for industrial use.