Vitamin B12 (cobalamin), as an essential cobalt-containing vitamin for the human body, boasts significant physiological functions and broad application in the pharmaceutical, animal feed, food, and cosmetic industries. It mainly exists in four forms: adenosylcobalamin, methylcobalamin, cyanocobalamin, and hydroxocobalamin (hydroxycobalamin). Cyanocobalamin, due to its chemical stability, serves as the primary industrial product form. Currently, microbial fermentation stands as the core method for industrial vitamin B12 production, with strains such as Pseudomonas denitrificans and Propionibacterium freudenreichii. Nevertheless, the production remains relatively low, which has become a key constraint for the industry’s development. This chapter highlights the principal innovations and notable accomplishments of the research team, led by Zhang Dawei from the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, in the domain of vitamin B12 biomanufacturing. By systematically reviewing the synthesis methods of vitamin B12, characteristics of industrial fermentation strains, metabolic engineering strategies, and fermentation technologies, this chapter clarifies the technical advantages and current development status of this field. Meanwhile, it analyzes the challenges encountered in the biomanufacturing process and the progress made in addressing them. Finally, it provides an overview of the global market pattern of vitamin B12 and the development trends of cost control. This comprehensive and in-depth review is expected to help readers gain a thorough and in-depth understanding of the vitamin B12 biomanufacturing.