Objective To establish a rapid method for obtaining Vav1 gene knockout stable cell lines. Methods Specific targeting vectors for the mouse Vav1 gene were constructed and transfected into the mouse melanoma cell line B16 using liposomes. GFP?positive single cells were sorted by flow cytometry. Genomic DNA was collected by directly lysing GFP?positive single cells and used for fluorescent PCR. The PCR product was loaded for capillary electrophoresis and the result were analyzed for rapid determination of the genotype. Results By using this method, a large number of GFP?positive single cells were obtained in a short time. Sixteen GFP?positive single cells were randomly selected and their genotypes were detected by fluorescence PCR; the result showed that the knockout efficiency was 87. 5%. Some of the single?cell genotypes were confirmed by Sanger sequencing and the result showed that the genotype result obtained by fluorescence PCR were completely correct. Conclusions The combination of the CRISPR/ Cas9 gene editing system, flow cytometry monoclonal sorting, fluorescence PCR, and high?throughput DNA sample processing technology enables a large number of Vav1 gene knockout stable monoclonal cells to be obtained in a short time in the mouse melanoma cell line B16.