The invention provides a construction method of a single enzyme digestion vector. The construction method comprises the following steps: 1) selecting single enzyme digestion sites; 2) introducing the enzyme digestion sites to the two ends of a target gene; 3) performing enzyme digestion and electrophoretic gel cut recovery on the vector and the target gene; 4) performing temperature gradient pretreatment on the mixed solution of the target gene and the vector; 5) connecting the vector and the target gene by use of solution I for connecting a T vector. The invention provides a new method for constructing the single enzyme digestion vector, and the enzyme digestion connection efficiency is greatly improved, and meanwhile, the connection time is greatly shortened. Due to the use of the method, the single enzyme digestion connection efficiency is remarkably improved, the connection time is only about 90min, the human and material resources are greatly saved and the operation difficulty of the single enzyme digestion is reduced.