The utilization and conversion of one-carbon (C1) feedstocks through biological processes have attracted much interest in building a carbon cycle economy. However, relevant research on the biosynthetic pathway of lactic acid (LA) from C1 compounds has not been reported. Herein, three de novo multienzyme synthetic pathways for optically pure and racemic LA from methanol were successfully engineered using a modularized construction and optimization approach involving enzyme screening, directed evolution, and improving reaction conditions. The general synthetic system achieved a productivity of 2.2–2.8 g/L LA with a maximum synthesis rate ranging from 9.6 to 15.6 g/L/day. Both L- and D-LA showed high stereoselectivity (>99% ee). This work provides an eco-friendly alternative approach for the production of LA, and it could contribute to carbon neutrality in the future.