Objective To establish miRNA knockout mouse model using CRISPR/Cas9 gene targeting technology. Methods According to the gene sequence of miRNA, we designed the primers of the gRNA targeting miR-301a (two targets) and obtained DNA template for in vitro transcription using PCR amplification; then we constructed Cas9 template for in vitro transcription, followed by in vitro transcription for gRNA of Cas9. In vitro transcribed gRNA/Cas9 mRNA was microinjected into the mouse zygote. T7E1 digestion and gene sequencing were used to detect and characterize the mutation of miRNA. Results PCR amplification, gel electrophoresis and gene sequencing proved that we had obtained the correct DNA template targeting miR-301a for in vitro transcription. By in vitro transcription we obtained gRNA/Cas9 mRNA, which was successfully microinjected into mouse zygote. The miR-301a mutants were detected by digestion with T7E1, and it was found that 7 of the 8 (87.5%) neonatal mice were found carrying mutations in miR-301a sites. Gene sequencing results showed that all mice had different levels of nucleotide insertion or deletion mutation, with the maximum number of 31 bases deletion found in No.4 mouse. Conclusion We have successfully established a mouse model with miR-301a gene knocked out, which lays a solid foundation for related future research.