In this study, cassava ‘Xinxuan 048’ was used as the material to observe and determine the morphological structure, endogenous hormones and the contents of carbon and nitrogen compounds in different stages of flower differentiation, and to conduct exogenous hormone regulation research to reveal the rules and the physiological mechanism of cassava flower differentiation. Cassava plants complete the whole process from flower bud differentiation to the end of flowering in 30 d, producing mainly two types of inflorescences: staminate and oligopistillate-monoecious. Both pistillate and staminate primordia were present in the same flower bud before sexual differentiation. The stamens gradually degraded in the female flowers 5 to 8 d after elongation of the inflorescence, while pistils gradually degraded and disappeared 5 to 7 d after elongation of the inflorescence, resulting in unisexual flowers. The contents of CTK and ETH were higher in female flowers than in male flowers in all the stages of differentiation; the GA3 content was higher in male flowers than in female flowers in the initial and middle stages of differentiation; the IAA content was significantly higher in male flowers than in female flowers in the middle stage of differentiation; the ABA content in male and female flowers was not of much difference in all the stages of differentiation; the contents of carbon and nitrogen were higher in male flower buds than in female flower buds, and the ratio of C/N was also higher in male flower buds than in female flower buds, especially in the initial stage of differentiation (5 d). The results of the study on the regulation of differentiation of male and female flowers by exogenous hormones showed that different concentrations of 6-BA and ETH had different promoting effects on females. The concentration of 6-BA and ETH at 60 mg/L and 1920 mg/L were the best, while GA3 promoted the formation of male flowers, with the optimal concentration being 40 mg/L. It can be concluded that the key period for female flower differentiation of cassava is 5 to 8 d after inflorescence elongation, and the key period for male flower differentiation is 5 to 7 d after inflorescence elongation. CKT and ETH can promote the female flower differentiation of cassava, and GA3, IAA and higher C/N ratio are more conducive to the male flower differentiation of cassava.