Abstract: Pi39 is a novel rice blast resistance gene located on rice chromosome 12. To clarify the resistant functions of Pi39, the knock-out vector of the candidate gene was constructed by the CRISPR/Cas9 system. Based on the analysis of the candidate gene, the two targeted site sequences were synthesized and inserted into the entry vector pENTR-sgRNA that was recombined into the expression vector Cas9 later. Firstly, the two target sites with PAM sequence were selected in the candidate gene, and then the two small target fragments were cloned into pENTR-sgRNA vector in turn. The results of colony PCR and Sanger sequencing indicated that the little purpose fragments inserted correctly. After the recombination of the entry vector and the expression vector, the positive clones were verified by colony PCR and restriction enzyme digestion and further confirmed by Sanger sequencing. Those results showed that the recombinant vector was successfully constructed. The knock-out vector was built of easy manipulation and time-saving by CRISPR/Cas9 system, which laid a foundation for further research on the function of Pi39 candidate gene.