〔Abstract〕 Objective To construct a conditional knockout target vector for the TDO2 gene in mice, and to lay a foundation for the establishment of conditional knockout mice of the TDO2 gene. Methods According to the principle of CRISPR/Cas9 technology, single guides RNA(sgRNA) and Donor vectors were designed and constructed. Exon 3 of the TDO2-201(ENSMUST00000029645.13) transcript was used as the knockout region, and a Loxp element was placed on each side of the target gene to establish a conditional gene target vector for conditional knockout of TDO2 exon 3. The CRISPR/Cas9 complex and Donor vector were microinjected into the fertilized eggs of C57BL/6 mice to obtain positive F0 mice. Then the positive F0 generation mice were mated with C57BL/6 mice to obtain F1 generation mice, and the F1 generation mice genotypes were identified by PCR and gene sequencing. Results The results showed that the constructed TDO2 gene conditional knockout vector met the design requirements, and B6/JGpt-TDO2em1Cflox/Gpt mice were successfully bred and identified. Conclusion The successful construction of the conditional knockout target vector of the TDO2 gene in mice builds the foundation for the further construction of TDO2 gene conditional knockout mice.