The aim of this study is to obtain the minimum concentration of glufosinate ammonium that can completely inhibit the growth of Cordyceps militaris and a method for quickly constructing the binary vector of bar gene. The concentration gradient method was used to study the inhibitory effect of glufosinate ammonium on the growth of C. militaris. Double-joint PCR （DJ-PCR） was used to construct the bar gene expression cassette. The T4 DNA ligase method and the homologous recombination method were used to insert the bar gene expression cassette into the T-DNA region of the binary vector pAg1-H3 to construct the binary vector pAg-Bar， and the ligation effects of the above two methods were compared. Agrobacterium tumefaciens-mediated transformation method was used to verify the effectiveness of the vector pAg-Bar and the function of the bar gene in C. militaris. The results showed that the minimum concentration of glufosinate ammonium that could completely inhibit the growth of C. militaris conidia was 400 μg/mL. The bar gene expression cassette could be effectively constructed by the DJ-PCR method. The binary vector pAg-Bar could be successfully constructed by the T4 DNA ligase method and the homologous recombination method. However， the homologous recombination method is simpler and more efficient than the T4 DNA ligase method. The bar gene expression cassette in the binary vector pAg-Bar could be successfully integrated into C. militaris genome by Agrobacterium tumefaciens-mediated transformation method， and the obtained C. militaris transformants were resistant to glufosinate ammonium. The method of constructing vectors established in this study is simple and efficient. It is suitable for constructing vectors with resistance gene， knock-out vectors， and overexpression vectors and so on. These results provide technical support for the identification of C. militaris gene function.