This study was purposed to explore the relationship between asparagine synthetase (AsnS) mRNA expression level and the sensitivity of leukemic cell lines to L-asparaginase. The AsnS mRNA expression level in 8 cell lines (Jurkat, HL-60, U937, NB4, THP-1, Namalwa, Karpas299 and K562) was determined by real-time quantitative PCR (RQ-PCR) based on fluorescence dye Eva Green before and after treatment with L-Asp, and the cell proliferation rates were analyzed by CCK-8 assay. The results showed that there was a significant disparity of AsnS expression level in 8 cell lines, and there were significant increases of AsnS expression level in cells co-cultured with L-Asp (p < 0.05). Of all these eight cell lines, cells sensitive to L-asparaginase had lower AsnS expression level and cells resistant to L-asparaginase had higher AsnS expression. U937 which was the most sensitive to L-asparaginase had the lowest AsnS expression level, while K562 was natural resistant to L-asparaginase and possessed of the highest AsnS level. It is concluded that the AsnS plays a critical role in regulating cellular biological behavior after depletion of asparagine, the AsnS mRNA expression level in cells reflects the sensitivity of cells to L-Asp. The results may imply the possibility for the use of L-asparaginase in leukemia with lower AsnS expression level.