L-serine is an important intermediate metabolite as a metabolic precursor to L-glycine and many other amino acids, nucleotides, choline, and phospholipids in organisms, and it has been widely used in medicine, food, cosmetics and other industries. The methods of producing L-serine include protein hydrolysis, chemical synthesis, enzymatic/microbial conversion and microbial fermentation, among which microbial fermentation has some advantages such as low cost of raw material, less environmental pollution, and high purity of product, etc. The strategies of metabolic engineering for improving L-serine production are reviewed, including the regulatory mechanisms and genetic modification of L-serine biosynthesis, as well the modified measures and subsequent effects. Furthermore, the prospect of L-serine breeding technologies is also discussed.