Bacterial artificial chromosome (BAC) library plays a pivotal role in genomics studies. A crucial step in BAC library construction is preparation of BAC vector DNA. Preparation of highly purified vector DNA is affected by a series of factors including digestion of restriction enzyme and dephosphorylation of linearized vector DNA. In our study, the BAC vector pECBAC1 was digested by the restriction enzyme of BamHI and dephosphorylated by HK phosphatase respectively. In order to improve the ligation capability of vector DNA, gel purification of linearized vector DNA was also conducted in our study. At the same time, we did a series of experiments to get high quality of vector DNA for construction of BAC library. They included the optimal concentration of restriction enzyme, optimal digestion time, the type of phosphatase and gel purification of linearized vector DNA.