Abstract: O-Methyltransferases (OMTs) are key enzymes involved in the methylation modification of benzylisoquinoline alkaloids (BIAs), and their functional studies contribute to elucidating the molecular mechanisms underlying BIA biosynthesis. In this study, a novel OMT gene was successfully cloned from Stephania cephalantha and subjected to bioinformatics analysis and in vitro functional characterization. The coding sequence (CDS) of ScOMT26 is 1 092 bp in length, encoding a protein of 363 amino acids with a molecular formula of C₁₈₂₇H₂₈₇₉N₄₆₇O₅₄₃S₁₈, classified as a stable hydrophobic protein. In vitro enzymatic assays demonstrated that ScOMT26 catalyzes the methylation of the C4′-hydroxyl group of coclaurine to produce 4′-methylcoclaurine. The optimal reaction conditions were identified as pH 7.0 in 50 mmol·L^-1 potassium phosphate buffer at 25 ℃. This study represents the first report of cloning and functional characterization of an OMT from S. cephalantha, providing critical insights into the role of OMT genes in the biosynthetic pathway of benzylisoquinoline alkaloids.