Enzymatic synthesis of L-4-nitrophenylserine from glycine and 4-nitrobenzaldehyde catalyzed by recombinant L-threonine aldolase in Escherichia coli BL21(DE3) with pGEX-KG plasmid was studied. Some factors influencing enzymatic reaction, such as temperature, pH, molar ratio of glycine to 4-nitrobenzaldehyde and substrate concentration were investigated. The optimal temperature and pH value were 45oC and 8.0. The optimal molar ratio of glycine to 4-nitrobenzaldehyde was 5:1, The optimal glycine concentration was 500 mmol/L. Under the optimal conditions, the bioconversion rate of 4-nitrobenzaldehyde reached 43% after 24 h. The concentration of L-4-nitrophenylserine reached 9.72 g/L, with yield of 35%.