Cunninghamia lanceolata (C. lanceolata) is the main fast-growing timber species in southern China. As an alternative to conventional lighting systems, LED has been demonstrated to be an artificial flexible lighting source for commercial micropropagation. The application of LED can provide rapid propagation of C. lanceolata in vitro culture. We applied two-factor randomized block design to study the effects of LED photoperiods and light qualities on the growth and chlorophyll fluorescence of C. lanceolata in vitro culture plantlets. In this study, plantlets were exposed to 20 μmol·m ·s irradiance for three photoperiods, 8, 16, and 24 h under the three composite lights, 88.9% red+ 11.1% blue (R/B), 80.0% red+ 10.0% blue+ 10.0% purple (R/B/P), 72.7% red+ 9.1% blue+ 9.1% purple+ 9.1% green (R/B/P/G), as well as white light (12.7% red+ 3.9% blue+ 83.4% green, W) as control. The results showed that: (1) Plant height, dry weight, rooting rate, average root number, length, surface area and volume, chlorophyll, and chlorophyll fluorescence parameters were significantly affected by photoperiods, light qualities and their interactions. (2) Plantlets subjected to photoperiod 16 h had longer root, higher height, rooting rate, root number, and the higher levels of chlorophyll, chlorophyll a/b, Y (II), qP, NPQ/4 and ETR compared to photoperiods 8 h and 24 h, while Fv/Fm during photoperiod 16 h was lower than 8 h and 24 h. Plantlets exposed to R/B/P/G generated more root and presented higher chlorophyll, Fv/Fo, Y (II), qP, and ETR than W during photoperiods 8 and 16 h. (3) Total chlorophyll content and ETR were significant correlated with rooting rate, root length and root volume, while Fv/Fm and ETR were significant correlated with plant height, average root number and root surface area. (4) 16-R/B/P/G is best for growing C. lanceolata plantlets in vitro. This study demonstrated the effectiveness of photoperiods and light qualities using LEDs for micropropagation of C. lanceolata. The best plantlets were harvested under 16-R/B/P/G treatment. And there was a correlation between the growth and the chlorophyll and chlorophyll fluorescence of their leaves under different photoperiod and light quality. These results can contribute to improve the micropropagation process of this species.