A high performance liquid chromatography-photodiode array detection-electrospray ionization mass spectrometry (HPLC-PDA-ESIMS) method has been developed for separation and identification of relative substances in hydroquinone bis(2-hydroxyethyl) ether (HQEE), a kind of polyurethane chain extender synthesized by hydroquinone (HQ) and ethylene oxide (EO). HQEE and all relative substances were baseline separated on a reverse-phase C 18 column with gradient elution of methanol and water. Of them, six relative substances were identified by ESIMS coupled with HPLC in positive ionization mode. The UV spectrum feature from online PDA and chromatographic retention behavior were adopted as evidence to conclude the rest relative substances. The results showed that EO combines with HQ in molar ratio of 1: 1-5: 1, and the tetra- (4: 1) and penta-substitution (5: 1) products are pairs of isomers, respectively. Therefore, the chromatographic fingerprint of industrial HQEE has been drawn on the basis of qualitative analysis of these products of different molar ratios.