The effects of different light intensities and qualities on the biomass, physiological parameters, and biochemical contents of hydroponically grown lettuce (Lactuca sativa L.) were evaluated, with the aim of obtaining better quality and higher yield, as well as saving energy in lettuce cultivation. Three different light qualities, provided by red (R), green (G), and blue (B) light-emitting diodes (LEDs), were used to produce six different combinations of illumination: A1: R:G:B = 7:0:3 [photosynthetic photon flux density (PPFD) = 150 μmol·m−2·s−1]; A2: R:G:B = 6:2:2 (150 μmol·m−2·s−1); A3: R:G:B = 7:0:3 (120 μmol·m−2·s−1); B1: R:G:B = 3:0:7 (150 μmol·m−2·s−1); B2: R:G:B = 2:2:6 (150 μmol·m−2·s−1); and B3: R:G:B = 3:0:7 (120 μmol·m−2·s−1), and the fluorescent lamp (FL) at 150 μmol·m−2·s−1 was used as the control (CK). In most cases, treatment A2 resulted in higher biomass attributes, whereas higher physiological parameters were observed in treatment B2. However, a greater shoot dry weight (SDW) was observed in treatment A1. No significant difference was detected in chlorophyll [Chl (a + b)] and carotenoid (CAR) contents among the different treatments. Soluble sugar content was found the highest in treatment A1, although it was not significant compared with that observed in treatment A2. Soluble protein content was higher in treatments with a higher component of blue light. Vitamin C content was found the highest in treatment B3 and the lowest in treatment A1, whereas malondialdehyde (MDA) content was the highest in CK and the lowest in treatments B1 and B2. These results indicated that appropriate ratio of red to blue light can effectively promote the accumulation of biochemical compounds in lettuce and that replacement of a certain portion of red light, blue light, or both with green light was more effective in promoting plant growth and quality.