Utilizing whole cell as biocatalyst instead of free or immobilized enzyme is a potential way to reduce the cost of catalyst in enzyme-catalyzed ?-D-2',3'- unsaturated-5 -fluorocytidine (D-D4FC). In this paper, D-D4FC was successfully synthesized by the whole cell of Lactobacillus helveticus (ATCC10697) for the first time. Several experimental parameters including reaction time, concentration of substrate, pH and the type of buffer were investigated; meanwhile a possible catalytic mechanism was discussed. Under the optimal reaction condition, about 50% yield of D-D4FC was obtained in 12.5 h and the cell of Lactobacillus helveticus showed good stability in the experiment. This method will be useful in the manufacture of important nucleoside analogues for anti-viral therapy.