[Objective] To study the anti-tumor activity of L-asparaginase II (ASP) of Escherichia coli Nissle1917. [Methods] L-AsparaginaseⅡ gene was amplified from the genome of E. coli Nissle1917 and inserted on the expression vector pET28a. The final expression vector pET28a-asp was transformed into E. coli BL21(DE3) for ASP expression. SDS-PAGE and LC-MS were used to determine the correct expression of ASP. After purification by nickel-affinity chromatography, the recombinant ASP was used to treat mouse 4T1 breast tumor cells, Hep-3B human hepatoma cells and Human Umbilical Vein Endothelial Cells (HUVEC). [Results] L-AsparaginaseⅡgene from E. coli Nissle1917 was successfully expressed in E. coli BL21(DE3) and identified by LC-MS. The purified L-asparaginaseⅡinhibited the growth of 4T1 and Hep-3B tumor cells while not HUVEC. [Conclusion] The L-asparaginaseⅡ of E. coli Nissle1917 was active on 4T1 and Hep-3B tumor cells while not on normal tissue cells. Our results will be helpful for the further study on the active mechanism of ASP and its application in solid tumors therapy.