Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland. However, purification of therapeutic proteins from transgenic milk are very important for productivity of recombinant protein. Development of a knock-in vector system is needed to improve production of therapeutic proteins. In this study, we are develop Knock-in vector to express human Erythropoietin protein (hEPO) using Gluthathione S-transferase (GST) fusion system on mouse β-casein exon 3 locus. The knock-in vector consisted of the 5 homologous arm (1.02 kb), GST, PreScission protease site, hEPO cDNA, BGH polyA signal, CMV-EGFP, and 3homologous arm(1.81 kb). The analysis of nucleotide and amino acid sequence revealed that GST-hEPO mRNA is probably translated with the mouse β-casein sequence and the β-casein-GST-hEPO fusion protein is probably secreted by ER-Golgi pathway. After that, the hEPO protein can be cleaved to remove the GST from the fusion protein by PreScission protease during purification of recombinant protein. This knock-in vector may help to create transgenic mouse expressing human Erythropoietin protein via the endogenous expression system of the mouse β-casein gene in the mammary gland.