Abstract: Carotenoid Cleavage Dioxygenase (CCD) serves as a key enzyme in the crocin biosynthetic pathway. To investigate the catalytic efficiency of different CCDs in tobacco chassis and to promote high-efficient heterologous synthesis of crocin in tobacco, six CCD genes related to crocin synthesis from five species were selected for transient expression in Nicotiana benthamiana, and the catalytic efficiency of different CCDs and the influence of heterologous synthesis of crocin on the carotenoid metabolic flux in tobacco were analyzed. The results showed that endogenous CCDs in tobacco could not catalyze the synthesis of crocin, while heterologous synthesis of crocin was achieved in the transgenic lines of CsCCD2L, BdCCD4.1, BoCCD4-3, and GjCCD4a. There were significant differences in the catalytic efficiency of different CCDs, with CsCCD2L and GjCCD4a exhibiting higher catalytic activities. Heterologous synthesis of crocin affected the metabolic flux of lycopene, β-carotene, and zeaxanthin in tobacco, suggesting that CCDs may have broader metabolic substrate specificity. Stable genetic materials for the heterologous synthesis of crocin were constructed by nuclear transformation, and optimization strategies for the heterologous synthesis of crocin in tobacco were clarified, which laid a crucial foundation for the development of a tobacco bioreactor for crocin production.