DNA recombinase Cre can recognize LoxP sites and result in recombination of DNA molecules. Recombination between two directly oriented LoxP sites excises the inserted DNA. And recombination between two circular DNA molecules, which contains a directly oriented LoxP site each one , generates a cointegrate. Based on these traits of Cre recombinase, a gene-transfer and gene sub-cloning system was constructed. gfp gene was excised from gene-donor vector pTLG and transferred directly to gene-receiving vector pET-LoxP mediated by Cre recombinase, completing the construction of pET-gfp very quickly and simply. Afterwards, gfp gene was expressed in Escherichia coli BL21(DE3) and generated visible green fluorescence colonies. It extremely simplified traditional courses of vector construction to only a reaction mediated by recombinase. Recombination ratios affected by circular gene-donor vector pTLG and linear pTLG were quantified and compared with. A good reference was provided for gene easy cloning or sub-cloning mediated by Cre recombinase.