As a substitute sweetener for sucrose, D-tagatose is widely used in products, such as health drinks, yogurt, fruit juices, baked goods, confectionery, and pharmaceutical preparations. In the fermentation process of L-AI produced by Lactobacillus plantarum, D-tagatose is produced through biotransformation and this study was based on the fermentation process of Lactobacillus plantarum WU14 producing l-AI to further research the biotransformation and separation process of D-tagatose. The kinetics of cell growth, substrate consumption, and l-arabinose isomerase formation were established by nonlinear fitting, and the fitting degrees were 0.996, 0.994, and 0.991, respectively, which could better reflect the change rule of D-tagatose biotransformation in the fermentation process of L. plantarum WU14. The separation process of D-tagatose was identified by decolorization, protein removal, desalination, and freeze drying, initially. Finally, the volume ratio of whole cell catalysts, D-galactose, and borate was 5:1:2 at 60 degrees C, pH 7.17 through borate complexation; then, after 24 hr of conversion, the yield of D-tagatose was 58 g/L.