In order to select the most suitable light quality ratio for the rooting of Cunninghamia lanceolata tissue cultured seedlings, we investigated the effects of different light quality treatments on the rooting rates of tissue cultured seedlings by using the superior clone of C. lanceolata ‘Yang 020’. And then, the differences of seedling growth, photosynthetic pigment content, chlorophyll fluorescence parameters, endogenous hormone content and chloroplast ultrastructure among the white light (control) treatment and the light quality treatments with highest and lowest rooting rates were analyzed, revealing the regulatory mechanism of rooting and growth of tissue cultured seedlings mediated by different light qualities, thus, providing theoretical basis for the factory production of C. lanceolata tissue culture seedlings. The results showed that: (1) under different light quality combination conditions, the rooting rate of seedlings treated with the combination of red and blue light, more specifically, red∶blue=3∶1 (R₃B₁) was the highest (71.11%), which is 45.61% higher than that of white light control, while the rooting rate of seedlings treated with blue light is the lowest (12.50%). (2) The biomass and plant height of seedlings under red and blue light treatment (R₃B₁) were higher than those of control and blue light treatments, and there was significant differences in plant height among different treatments (P＜0.05). (3) The photosynthetic pigment content and PSⅡ potential photochemical efficiency F_v/F_o value of seedlings in red and blue light (R₃B₁) treatment were significantly higher than control and blue light treatments, suggesting leaves may have strong light source capturing ability and light energy utilization ability. (4) The ultrastructure of chloroplast results showed that the number and size of chloroplast starch grains in the leaves of seedling treated with the combination of red and blue light (R₃B₁) was small and the structure of chloroplast matrix and grana lamella was clear, which might favorable for the output of assimilation products. (5) The higher rooting rate of the seedlings treated with the combination of red and blue light (R₃B₁) was associated with higher auxin (IAA), lower cytokinin (CTK) content and higher IAA/CTK ratio in its shoots, while the higher plant height was associated with higher gibberelins (GA), IAA contents, IAA/ABA and GA/ABA ratios, and lower ABA content in its shoots.