D-Tagatose is an important rare ketohexose. Its sweetness is 92% of sucrose, and its calories are only one third of sucrose. D-Tagatose was approved as a new raw material for safe food by the America's Food and Drug Administration and China's Health and Family Planning Commission in 2001 and 2014, respectively. D-Tagatose has the functions of lowering blood sugar, reducing obesity, improving gut microbiota, anti-caries, anti-oxidation and so on. D-tagatose, as an ideal functional sweetener, has extensive application value. However, the production cost and yield of D-tagatose have always been an important reason limiting the development and application of D-tagatose. The methods for D-tagatose synthesis mainly include chemical route and biological conversion. Although it is cost-effective to produce D-tagatose by chemical approach, it requires high temperature and pressure in the production process, and is easy to generate miscellaneous sugars such as sorbose and mannose, which is not conducive to separation and purification. In order to overcome these shortcomings, the biological conversion of D-tagatose has received extensive attention. Therefore, the research progress, advantages and disadvantages of D-tagatose biosynthesis from different substrates such as hexoses (galactitol, D-galactose and D-fructose), lactose, polysaccharide (maltodextrin and starch) were systematically reviewed, and the research trends in the future were also prospected, which provided reference for the research and production of D-tagatose.