In this study, the cucumber ‘Jinchun No. 2’ cDNA was used as the template, and the key enzyme gene (CsPAO) for chlorophyll degradation (pheophorbide a oxygenase, PAO) of cucumber was cloned by RTPCR method, and the subcellular location was observed and realtime fluorescence was used. Quantitative PCR technology and bioinformatics technology analyzed the expression pattern of CsPAO gene and the characteristics of its encoded protein. The results showed that: (1) CsPAO encodes 545 amino acids, the theoretical isoelectric point is 6.09, and the relative molecular mass of the protein is 61.02 kD. Protein prediction found that cucumber CsPAO is an unstable protein with two protein binding sites and a transmembrane phenomenon. (2) Fluorescence quantitative PCR results show that CsPAO responds to salicylic acid (SA), jasmonic acid (JA) and gibberellin (GA₃), the expression of CsPAO gene under high temperature (42 ℃) and low temperature (4 ℃) treatments, the amount increased significantly and the expression reached the highest, but the dark treatment had no effect on the expression of CsPAO gene; the expression in flowers of cucumber was significantly higher than that of roots, stems, leaves, calyx, whiskers and fruit. (3) The subcellular localization results show that the CsPAO protein is localized in the chloroplast. (4) Phylogenetic tree analysis shows that cucumber CsPAO is closely related to bitter gourd, summer squash, pumpkin and winter squash. The results of this study laid the foundation for further revealing the molecular mechanism of cucumber chlorophyll degradation.