Tetrahydroprotoberberines (THPBs), mainly sourced from the medicinal plants of genus Stephania and Corydalis, exhibit a variety of pharmacological activities. Plant-sourced THPBs are limited by low yields, and their chemical synthesis faces high cost, low yield, and stereochemical challenges. Since the biosynthesis of THPBs has not yet been fully established, further investigation into their pharmaceutical utility is limited by the product quantity. Here, by reconstructing tailor-made biosynthetic pathways, we provide an alternative strategy for the production of THPBs and related intermediates in Escherichia coli (E. coli). Firstly, the major obstacle in the reconstruction of the THPBs biosynthetic pathway, berberine bridge enzyme (BBE), a rate-limiting enzyme was functionally expressed in E. coli using several new strategies. Next, the corresponding biosynthetic pathway modules for the biosynthesis of (S)-scoulerine (compound 3), (S)-tetrahydropalmatine (compound 5), (S)-corydalmine (compound 6), and their intermediates were reconstituted. We achieved the biosynthesis of compounds 3, 5, and 6 and their intermediates in the corresponding engineered E. coli strain. Furthermore, we optimized culture conditions to raise the yield of compound 5 to 1.19 mg L⁻¹ in a flask culture using the engineered strain. Our work provides a green solution for the production of high-value THPBs without environmental consequences.