【Objective】 Chlorophyllide a oxygenase (CAO) is a key enzyme in the formation of chlorophyll b. To investigate the regulation function of the CAO gene in the leaf color variation of Cymbidium hybrid, and to provide an important basis for studying the formation mechanism of leaf art. 【Methods】 ChCAO gene was cloned from leaves of Cymbidium hybrid by RACE and RT-PCR methods. ChCAO was analyzed for structure characteristics, physicochemical properties, sequence alignment and phylogenetic relationship. The expression characteristics of ChCAO in different tissues and leaves of K21-3 were analyzed by qRT-PCR. 【Results】The results showed that ChCAO coding region was 1 608 bp long, which encoding 535 amino acids. ChCAO was closely related to Cymbidium sinense, which also belonged to genus Cymbidium. The qRT-PCR results reflected the tissue-specific in the expression of ChCAO, with the highest relative expression in leaves and the lowest in roots. Among the different varieties, the expression level of ChCAO in K21 leaves were significantly higher than that of K21-3. The VIGS silencing vector of this gene was constructed and transferred to tobacco, it was found that the viral vector successfully invaded the body of tobacco, the old leaves of tobacco were in a yellow state, and the chlorophyll content in the old leaves and the relative expression of the ChCAO gene were also significantly reduced. It is speculated that the silent expression of ChCAO gene may lead to the reduction of chlorophyll content and the yellowing of leaves, which preliminarily clarifies the function of the ChCAO gene in Cymbidium hybrid.