Psoralea corylifolia L., a widely used traditional Chinese medicine, has recently demonstrated therapeutic potential in the treatment of liver cancer. However, rigorous evaluations regarding its clinical efficacy and quality control remain insufficient. To address this issue, this study selected 10 batches of Psoralea corylifolia L. from different habitats as research objects. Firstly, high-performance liquid chromatography (HPLC) was employed to optimize the chromatographic conditions, and based on this, the chemical fingerprints of the 10 batches of medicinal materials were constructed to characterize their chemical composition profiles. Meanwhile, the IC₅₀ values of medicinal extracts from each batch against Hepa1-6 liver cancer cells were determined; using spectrum-effect correlation analysis, potential anti-liver cancer active components were preliminarily screened, and their activities were further validated by cell experiments. Finally, an effective-constituents index (ECI) was established for the 10 batches of Psoralea corylifolia L. and applied to its quality evaluation. The results showed that although the similarity of the chemical fingerprints of the 10 batches of Psoralea corylifolia L. exceeded 0.95, indicating a relatively consistent overall chemical composition, the content of common components varied significantly among different batches. In terms of anti-liver cancer activity, the IC₅₀ values of the 10 batches of medicinal materials against Hepa1-6 cells ranged from 34.54 μmol·L^-1 (minimum) to 85.04 μmol·L^-1 (maximum), showing obvious differences in activity, which further confirmed the necessity of establishing a more targeted quality evaluation system. Through spectrum-effect relationship analysis and in vitro cell activity verification, it was confirmed that isobavachin, neobavaisoflavone, bavachin, psoralidin, isobavachalcone, bavachinin and bakuchiol are the main active components responsible for the anti-liver cancer effect of Psoralea corylifolia L. Notably, psoralen and isopsoralen, which are specified as index components in the Pharmacopoeia, showed no inhibitory activity against Hepa1-6 liver cancer cells in this study. In addition, the constructed ECI could classify the 13 batches of Psoralea corylifolia L. into three quality grades, and this classification result was consistent with the results of linear discriminant analysis, indicating good reliability of the ECI. More importantly, the correlation coefficient between the ECI of Psoralea corylifolia L. and its anti-liver cancer activity reached as high as 0.957, which fully proved that the ECI can effectively reflect the anti-liver cancer efficacy of Psoralea corylifolia L. In conclusion, the ECI established in this study can be used for the quality evaluation of the anti-liver cancer effect of Psoralea corylifolia L. It not only provides a new method for improving the quality control standard of Psoralea corylifolia L. but also offers a valuable reference for the quality control and efficacy evaluation of other anti-tumor traditional Chinese medicines.