Abstract: ABSTRACT: 【Objective】 To clone the 3-O-glucosyltransferase gene (3GT), a key enzyme in the saponin biosynthesis pathway, and explore the biosynthetic mechanism of the main active saponins in Conyza blinii. 【Methods】 Based on previous research and Conyza blinii transcriptome data, the 3GT gene was cloned using PCR, and its molecular characteristics were analyzed using bioinformatics tools, while molecular docking was employed to predict its substrate specificity. 【Results】 The full-length cDNA of Conyza blinii 3GT was 1470 bp, encoding 489 amino acids with a predicted molecular weight of 54.88 kDa, an isoelectric point of 5.50, an instability index (II) of 43.12, and a hydrophilic coefficient (GRAVY) of -0.185, indicating that CbUGT is an unstable hydrophilic protein. BLAST analysis revealed that CbUGT contains the glycosyltransferase family 1 domain and a GTB topology-related domain, lacks signal peptides and transmembrane regions, and is predicted to localize to the cell membrane. Secondary structure analysis showed a predominance of α-helices, and tertiary structure prediction suggested a monomeric form. Molecular docking simulations indicated binding affinity for both glucose and oleanolic acid, consistent with structural analysis. Phylogenetic analysis grouped CbUGT with Arabidopsis thaliana UGT73C1 (Class D). 【Conclusion】 CbUGT is a typical plant 3-O-glucosyltransferase gene involved in the transfer of glucose to the C3 position of saponins in Conyza blinii.