Polygonatum cyrtonema Hua was used as the test material to establish the HPLC fingerprint and extract its characteristic fingerprint information. Similarity evaluation, cluster analysis, and orthogonal partial least squares discriminant analysis (OPLS-DA) methods were employed to comprehensively analyze the irradiation of Polygonatum cyrtonema Hua with different doses of electron beams and screen for differential marker substances. The antioxidant activity of Polygonatum cyrtonema Hua was evaluated through DPPH and ABTS+ free radical scavenging rates. The spectrum-effect relationship between the fingerprint and antioxidant activity was explored using partial least squares regression analysis (PLSR).The results indicated that the similarity of HPLC fingerprints of Polygonatum cyrtonema Hua ranged from 0.972 to 0.994 at different doses, with 19 common peaks identified. Peak 2 was identified as rutin, peak 9 as quercetin, and peak 10 as liquiritin. OPLS-DA identified nine landmark differential substances before and after irradiation, including rutin, liquiritin, and quercetin. Spectrum-effect analysis demonstrated that 13 components were positively correlated with antioxidant activity, with Peak 1, Peak 6, and rutin identified as strong antioxidant substances in Polygonatum cyrtonema Hua. This study reflected the differences in composition and free radical scavenging ability with different doses of electron beam irradiation of Polygonatum cyrtonema Hua, providing effective data for quality monitoring of electron beam irradiation of Polygonatum cyrtonema Hua.