Ultra-performance liquid chromatography (UPLC) fingerprint of Sophorae Flavescentis Radix was established, and its anti-tumor spectrum-effect relationship was explored to preliminarily elucidate the material basis of its anti-tumor effect. Seventeen batches of Sophorae Flavescentis Radix were analyzed by UPLC to establish a fingerprint profile and, by alignment with the control herbs, a total of 16 common peaks were identified. Furthermore eleven compounds were identified by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and the reference substance. Human non-small cell lung cancer A549 cells were used as the test tumor cell line, and the CCK-8 method was employed to detect the anti-tumor activity of Sophorae Flavescentis Radix from various origins. The half maximal inhibitory concentration was used as the index; multiple chromatographic peaks were found to be closely related to anti-tumor activity using grey correlation degree and partial least squares discriminant analysis. By comprehensively taking into account three indicators, namely the positive or negative correlation coefficient of the regression equation, the variable importance in projection value, and the grey relational coefficient, it was preliminarily determined that peaks 2 (oxymatrine), 3 (oxysophocarpine), 4 (9α-Hydroxymatrine), 6, 8 (matrine), 9 (sophoramine), 13, 14, 15 (naringenin) and 16 (kurarinone) are the primary material basis for the anti-tumor effect of Sophorae Flavescentis Radix. Additionally, the validation experiments on the anti-tumor activities of the active ingredients demonstrated a concentration-dependent inhibitory effect of kurarinone, oxymatrine, oxysophocarpine, and matrine on the growth of A549 cells. This study not only established the UPLC fingerprint of Sophorae Flavescentis Radix but also provided a preliminary understanding of the material basis of its anti-tumor efficacy, providing a reference for further research on the material basis of its pharmacological effects and quality evaluation.