Benzylisoquinoline alkaloids (BIAs) constitute a vast and diverse class of plant-derived secondary metabolites, renowned for their potent therapeutic activities. O-methylation, catalyzed by O-methyltransferases (OMTs), is a prevalent feature of BIA molecules, which is intrinsically related to their bioactivities. Herein, we described the identification and characterization of two functionally identical but phylogenetically different BIA 7OMTs, SyOMT4 and SyOMT5, from the transcriptomic data of Stephania yunnanensis-a well-known anti-COVID-19 drug cepharanthine producing plant. Through multiple sequence alignment, structural modeling as well as site-directed mutagenesis, three residues (R143/L156/H163 in SyOMT4, and F154/F167/G174 in SyOMT5) were identified to be involved in substrate binding micro-environment. Their manipulation led to redirection of the catalytic regioselectivities of SyOMT4 and SyOMT5. SyOMT4 was adeptly transformed into a highly specific BIA 6OMT, namely SyOMT4-WFE, and unexpectedly SyOMT5 was engineered into BIA NMTs, designated SyOMT5-WFE and SyOMT5-WFD). This study shed light on protein engineering to expand the functional repertoire of BIA OMTs by manipulating their substrate binding micro-environments, enlarging the enzymatic toolkit for the production of medicinally important BIAs by using synthetic biology and metabolic engineering strategies.