Objective: Establishing the F9 gene knockout cell lines of porcine fetal fibroblasts (PFFs) of Bama minipig by CRISPR/Cas9 gene-editing technology provide raw materials for the subsequent construction of Bama minipig models of hemophilia B. Methods: Firstly, we analyzed the homology of human and pig F9 gene and the similarity of the secondary and tertiary structure of human and pig FⅨ by bioinformatics method. Secondly, we selected the second exon of pig F9 gene as the targeting region and used online tools (http://www.e-crisp.org/E-CRISP/designcrispr.html) to design gRNAs. The pX330 plasmid is used as the backbone to construct targeting vectors, which were then co-transfected with a G418 resistant plasmid into the PFFs of wild Bama miniature pigs. The G418 resistant single-cell colonies were obtained and sequenced. Results: Bioinformatics analysis showed that the F9 gene of human and pig had a close phylogenetic distance. The similarity value of the amino acid sequence of FIX and the RMSD value of the three-dimensional structure was 83.33% and 0.149, respectively. A total of 55 resistant single-cell colonies were obtained by G418 screening. Twenty-five single-cell colonies bearing mutations in the F9 gene targeting region have been identified by sanger sequencing, indicating the knockout efficiency was 45.5%. Conclusion: The human and pig F9 genes have a high degree of homology. The constructed Cas9/sgRNA expression vectors can efficiently induce mutagenesis in the pig F9 gene. The F9 gene knockout PPFs lays an essential foundation for the construction of Bama minipig model of hemophilia B.