sp. d-tagatose 3-epimerase (sDTE) catalyzes the conversion of d-tagatose to d-sorbose. It also recognizes d-fructose as a substrate for d-allulose production. The optimal temperature and pH of the purified sDTE was 50 °C and 8.0, respectively. Based on the sDTE homologous model, Glu154, Asp187, Gln213, and Glu248, form a hydrogen bond network with the active-site Mn and constitute the catalytic tetrad. The amino acid residues around O-1, -2, and -3 atoms of the substrates (d-tagatose/d-fructose) are strictly conserved and thus likely regulate the catalytic reaction. However, the residues at O-4, -5, and -6, being responsible for the substrate-binding, are different. In particular, Arg65 and Met9 were found to form a unique interaction with O-4 of d-fructose and d-tagatose. The whole cells with recombinant sDTE showed a higher bioconversion rate of 42.5% in a fed-batch bioconversion using d-fructose as a substrate, corresponding to a production of 476 g Ld-allulose. These results suggest that sDTE is a potential industrial biocatalyst for the production of d-allulose in fed-batch mode.