Recombinant Escherichia coli pUDP, which overexpressed uridine phosphorylase (UPase), was constructed. 0.5 mmol.L-1 lactose had a similar induction effect as the commonly used inducer IPTG during 2.5-5.5 h of cell growth. The lactose-induced UPase was stable at 50 degrees C. Wet cells of pUDP was used as catalyst to biosynthesize 5-fluorouridine from 30 mmol.L-1 uridine and 5-fluorouracil in phosphate buffer (pH 7.0) catalyzed at 50 degrees C for 1.5 h and the yield of 5-fluorouridine was higher than 68%. Under the optimum reaction conditions for production of 5-fluorouridine, 5-methyluridine and azauridine were synthesized from uridine by pUDP, the yield was 61.7% and 47.2% respectively. Deoxynucleosides were also synthesized by pUDP, but the yield was only about 20%.