In this study, a UVB photoreceptor gene (MsUVR8) of alfalfa was cloned by RACE amplification. On the basis of bioinformatics analysis, Agrobacterium mediated method was used to obtain the overexpressed callus of MsUVR8. The contents of flavonoids, flavonols, anthocyanins, hydrogen peroxide (H₂O₂), superoxide anion (O₂^-·) and UVB signaling pathway related genes in MsUVR8 overexpressed callus and wild type after UVB radiation treatment were detected and analyzed. The purpose of this study is to supply a theoretical foundation for revealing the molecular mechanism of plant response to UVB stress. The results showed that: (1) the CDS sequence of MsUVR8 in alfalfa was 834 bp, and the similarity between MsUVR8 and MtUVR8 gene of Medicago truncatula was more than 95%. The MsUVR8 protein formed an incomplete βfolded structure, and phylogenetic analysis showed that it belongs to the same branch as chickpea. (2) It was found that the content of flavonoids in MsUVR8 overexpressed callus (UVR8OE) of alfalfa was significantly higher than that of wildtype callus (WT), and the content of flavonoids in UVR8OE after UVB radiation was further significantly higher than that of WT. (3) DPBA fluorescence labeling showed that UVB radiation greatly promoted the synthesis of flavonols, and UVR8OE had the highest flavonols content after UVB radiation. (4) DAB and NBT staining showed that the accumulation of reactive oxygen species (H₂O₂ and O₂^-·) in WT increased after UVB treatment, but there was no significant difference between UVBtreated and untreated UVR8OE, indicating that MsUVR8 could enhance the antioxidant capacity of plant tissues and cells and reduce the oxidative damage caused by UVB stress. (5) After UVB irradiation, the expression of PAL, CHS and FLS in WT was activated and significantly increased, and the expression of four genes in UVR8OE reached the maximum, which was significantly increased compared with the other three treatment groups. The results showed that alfalfa MsUVR8 activated by UVB promoted the expression of genes related to flavonoid synthesis and activated the activities of key enzymes in flavonoid synthesis, thus improving the efficiency of flavonoid synthesis and enhancing the antioxidant capacity of plant callus under UVB stress.