Abstract: Human milk oligosaccharides (HMOs) are key components of breast milk, playing a vital role in fostering the healthy development of infants and young children. Among the main HMOs components, 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) exhibit considerable benefits in terms of inhibiting pathogenic infections, regulating gut microbiota, and enhancing immunity. In recent years, the synthesis of 2′-FL and 3-FL has emerged as a prominent area of research in the field. Despite the potential advantages of chemical or enzymatic synthesis of 2′-FL and 3-FL, these methods are hindered by their inherent limitations, including intricate reaction procedures, high costs, and poor yield, which are not conducive to meeting the demand of the market. Therefore, more efficient microbial methods have been developed and applied in the commercial production of 2′-FL and 3-FL. This article systematically outlines the methods for preparing both 2′-FL and 3-FL, thoroughly expounds upon the engineering strategies for microbial synthesis of 2′-FL and 3-FL, and provides a compelling framework for further microbial production exploration of 2′-FL and 3-FL.