Lettuce was grown in the fully artificial light plant factory, where adjustable red and blue LED panels were used as the sole light source for lettuce growth. Red and blue light with different alternating frequencies were provided to test plant responses to the alternating red and blue lights. Meanwhile, concurrent red and blue light treatments were set as controls. Results were analyzed in terms of the growth dynamics, and the accumulation of biomass, photosynthesis pigments, soluble sugar, crude protein, vitamin C contents as well as nitrate content in lettuce. The results showed that based on the same energy consumption, alternating red and blue lights with the frequency of one time (R/B1) in a 16h period promoted the accumulation of biomass, soluble sugar and crude protein contents, while alternating red and blue lights with the frequency of four times (R/B4) in a 16h period enhanced the vitamin C content and decreased nitrate content of lettuce. Among all the treatments, the highest chlorophyll and carotenoid contents were both detected under R/B1 or R/B4 treatments, no significant difference existed between the two treatments for the pigment content. Therefore, the focal point was the comparison of red and blue lights provided at the same time and those provided separately with different alternating intervals based on the same daily light integral. The goal was to determine the effects of different radiation modes of red and blue LED lights on the growth and quality of lettuce. The alternating modes would provide methods for deeply studying the relationship of red and blue lights when acting on plants. Meanwhile, the selection of light formula based on the same energy consumption was more acceptable in practical production.