v-Src is a non-receptor protein tyrosine kinase, which is involved in many signal transduction pathways and closely related to the activation and/or development of cancers. Here we presentedthe expression, purification and initial characterization of GST-fusion v-Src in plsmid pLysS from abaterial expression system. Different culture conditions were examined in the indution of IPTG andGSH affinity chromatography was used to purified v-Src protein. With the versatile sub- strate poly-(Glu:Tyr) 4:1, ELISA was employed to ditermine the phosphorylation kinase activity of GST-fusedv-Src. This strategy seems to be more promise than the insect cell system or other eukaryotic systemsemployed in earlier Src expression.