Benzylisoquinoline alkaloids (BIAs) are the primary active components of Stephania tetrandra. However, the molecular mechanisms underlying BIA biosynthesis in S. tetrandra remain poorly understood. Here, we reported the isolation and characterization of a novel O-methyltransferase, designated as St7OMT1, from S. tetrandra. St7OMT1 exhibited strict substrate specificity and regioselectivity, catalyzing the conversion of (S)-coclaurine to norarmepavine at the C7 position. Optimal enzymatic activity of St7OMT1 was detected at 30 °C and pH 6.0 in sodium citrate buffer, with kinetic parameters of K