A method of improving the recombination efficiency of larger plasmid DNA is introduced. The major manipulation of this method is that the ligation reaction between insertor and vector is separated from cycle reaction of ligation, and they carry out in different reaction condition. Using this method, the efficiency of the recombination between 7.65 kb vector and 1.47 kb insertor is improved obviously. It is a good method for recombination of larger plasmid DNA.