Abstract: To clarify the effects of long-day photoperiod on flower bud differentiation in Paulownia fortunei, this study used five-month-old seedlings as materials and applied 16 h and 24 h long-day photoperiod treatments. The flower bud differentiation rate, external and internal morphology of flower buds at different differentiation stages, soluble sugar (SS), soluble protein (SP), and endogenous hormones were quantitatively analyzed in P. fortunei. The results showed that the number of seedlings undergoing flower bud differentiation was the same under both treatments, but the 24 h treatment significantly increased the flower bud differentiation rate of P. fortunei by 13.34% (p<0.05). The flower bud differentiation process of P. fortunei could be divided into four stages from S₀ (undifferentiated) to S₃ (late differentiation), and all stages under the 24 h treatment occurred approximately two weeks earlier than those under the 16 h treatment. Throughout the differentiation process, the soluble sugar content in flower buds of P. fortunei under the 24 h treatment was significantly higher than that under the 16 h treatment (p<0.05), whereas no significant difference was observed in soluble protein content. The 24 h treatment significantly increased GA₃, ZR, and ABA contents and decreased IAA content in flower buds of P. fortunei; meanwhile, it significantly increased the ratios of GA₃/IAA, ZR/IAA, and ABA/GA₃, and these hormonal ratios exhibited clear stage-specific dynamics during flower bud differentiation. Specifically, high GA₃/IAA and ZR/IAA ratios during S₀～S₁ were favorable for flower bud initiation in P. fortunei; an increased ZR/GA₃ ratio during S₂ promoted the formation of floral organ primordia; and an elevated ABA/GA₃ ratio during S₃ was conducive to flower bud maturation. Overall, long-day photoperiod may promote flower bud differentiation and development, shorten the differentiation process, and advance flowering of P. fortunei by enhancing carbohydrate accumulation and coordinating hormonal balance.