Benzylisoquinoline alkaloids (BIAs) are an important class of plant secondary metabolites with a variety of pharmacological activities. Although they are widely used, traditionally these compounds are extracted from natural sources because their structure is too complicated to achieve economically feasible chemical synthesis. Thus, microbial biosynthesis of BIAs is expected to reduce dependence on natural extracts. (S)‐Reticuline is an important precursor for BIAs biosynthesis. Therefore, it is an attractive engineering target. In this study, we reported the development of a novel (S)‐reticuline biosynthetic pathway based on 4‐hydroxyphenylacetate 3‐hydroxylase (HpaBC) in Escherichia coli. Then, we further improved the (S)‐reticuline production to 307 ± 26.8 mg/L by increasing the availability of the precursor 3, 4‐dihydroxyphenylacetaldehyde. The E. coli cell factory developed in this study can be used as a potential platform for further efficient biosynthesis of BIAs derivatives.