PNP oxidase is an important invertase in the VB₆ metabolic pathway. In this study, the tea tree ‘Longjing 43’ was used as the material, and the PNP oxidase gene was cloned by RTPCR. The prokaryotic expression vector was constructed by using pET22b(+) as a vector, and induced expression by IPTG for functional identification; Fluorescence quantitative PCR was used to analyze the differential expression of CsPNPO gene in different tissues of tea plants and its expression under low temperature and drought stresses, which laid a foundation for further analysis of physiological and biochemical functions of tea tree VB₆. The results showed that: (1) the coding frame length of CsPNPO was 1 503 bp, and it was predicted to encode a 501 amino acid protein. The molecular weight of CsPNPO protein was 48.5 kD, and the theoretical isoelectric was 5.82. The CsPNPO protein contains no signal peptide and is a nonsecreted hydrophilic protein, and localized to chloroplasts. Amino acid sequence analysis revealed that it has a chloroplast transit peptide region, a YjeFN functional domain, and a PNP oxidase functional domain. (2) The prokaryotic expression vector pET22b(+)CsPNPO was successfully constructed, and the recombinant protein had strong PNP oxidase activity at pH 8.5 and 37 ℃. (3) Fluorescence quantitative PCR detection shows that the expression level of CsPNPO gene in tea leaves was the highest, followed by stems, and the expression in roots was only one tenth of that of leaves, indicating that CsPNPO gene has tissue expression specificity; The expression level of CsPNPO gene decreased significantly under low temperature and drought conditions. It is speculated that CsPNPO gene may be involved in the stress response of tea tree to low temperature and drought.