Due to the complicated metabolic and regulatory networks of l-serine biosynthesis and degradation, microbial cell factories for l-serine production using non-model microorganisms have not been reported. In this study, a combination of synthetic biology and process optimization were applied in an ethanologenic bacterium Zymomonas mobilis for l-serine production. By blocking the degradation pathway while introducing an exporter EceamA from E. coli, l-serine titer in recombinant Z. mobilis was increased from 15.30 mg/L to 62.67 mg/L. It was further increased to 260.33 mg/L after enhancing the l-serine biosynthesis pathway. Then, 536.70 mg/L l-serine was achieved by removing feedback inhibition with a SerA mutant, and an elevated titer of 687.67 mg/L was further obtained through increasing serB copies while enhancing the precursors. Finally, 855.66 mg/L l-serine can be accumulated with the supplementation of the glutamate precursor. This work thus not only constructed an l-serine producer to help understand the bottlenecks limiting l-serine production in Z. mobilis for further improvement, but also provides guidance on engineering non-model microbes to produce biochemicals with complicated pathways such as amino acids or terpenoids.