To evaluate the xanthine oxidase inhibitory activity of the chemical constituents of Ligustrum lucidum in vitro, the spectrum-effect relationship was investigated. The HPLC fingerprint was established by ultraviolet spectrophotometry, and the xanthine oxidase inhibitory activity was tested in vitro by a high-throughput screening method. Cluster analysis, principal component analysis, gray correlation analysis and partial least squares regression were used to explore the spectrum-effect relationships. Sixty batches of Ligustrum lucidum were collected from 16 provinces for testing. The results revealed differences among the batches of medicinal materials, and the similarity score was between 0.635 and 0.968. Thirty-three characteristic peaks (1-33) were calibrated by fingerprint evaluation software for traditional Chinese medicine. The spectrum-effect relationship study further revealed that the contents of peaks 1, 2, 4, 5, 6, 7, 14, 17, 25, 28, 31 and 33, which are potentially critical ingredients for quality control of Ligustrum lucidum fruit, were highly correlated with the inhibition of xanthine oxidase activity. This article is protected by copyright. All rights reserved.