OBJECTIVE：To establish HPLC fingerprint of Humulus lupulus ，and to investigate its correlation with the antioxidant activity. METHODS ：The determination was performed on Diamonsil C 18 column with mobile phase consisted of 0.1％ phosphoric acid solution-acetonitrile （gradient elution ）at the flow rate of 1.0 mL/min. The column temperature was 30 ℃，and detection wavelength was set at 358 nm，with sample size of 2 μL. Using xanthohumol as reference，HPLC fingerprints of 11 batches of H. lupulus were determined. The similarity of 11 batches of samples was evaluated by TCM Chromatographic Fingerprint Similarity Evaluation System （2012 edition）to confirm common peaks. Using scavenging rate of DPPH and ABTS free radical as pharmacodynamic indicators of antioxidant effects ，SIMCA 14.1 analysis software was used for PLSR to establish the spectra-effect relationship ，and validation test of in vitro anti-oxidation was carried out. RESULTS ：There were 19 common peaks in HPLC fingerprints of 11 batches of sample ，the similarity of which was higher than 0.830. Seven components were identified as xanthohumol，cohumulone，humulone，adhumulone，colupulone，lupulus and adlupulus. Eleven batches of H. lupulus had the ability to scavenge DPPH and ABTS free radicals. The spectrum-effect relationship showed that xanthohumol ，humulone and colupulone peaks were positively associated with its anti-oxidant ability ，and variable projection value was greater than 1. In vitro antioxidant results showed that scavenging effect of 0.1 mg/mL xanthohumol to DPPH free radicals was similar to that of 0.01 mg/mL vitamine C ，but scavenging effect of 10.0 mg/mL xanthohumol to ABTS free radical was less than that of 0.1 mg/mL vitamin C. CONCLUSIONS：Established HPLC fingerprint can be used for the quality evaluation of H. lupulus ；xanthohumol，humulone and colupulone are the main material basis for the antioxidant effect of H. lupulus