Lactobacilli have been widely concerned for decades. Bacteria of the genus Lactobacillus have been commonly employed in fermented food to improve the appearance, smell, and taste of food or prolong its shelf-life. They comprise 261 species (by March 2020) that are highly diverse at the phenotypic, ecological, and genotypic levels. Some Lactobacilli strains have been documented to be essential probiotics, which are defined as a group of living microorganisms that are beneficial to the health of the host when ingested in sufficiency. However, the characterization, high-density fermentation, and the production of a directed vat set (DVS) starter of Lactobacilli strains used in the food industry have not been systematically reported. This paper mainly focuses on reviewing Lactobacilli as functional starter cultures in the food industry, including different molecular techniques for identification at the species and strain levels, methods for evaluating Lactobacilli properties, enhancing their performance and improving the cell density of Lactobacilli, and the production techniques of DVS starter of Lactobacilli strains. Moreover, this review further discussed the existing problems and future development prospects of Lactobacilli in the food industry. The viability and stability of Lactobacilli in the food industry and gastrointestinal environment are critical challenges at the industrial scale. The new production equipment and technology of DVS starter of Lactobacilli strains will have the potential for large-scale application, for example, developing low-temperature spray drying, freezing granulation drying, and spray freeze-drying.