The use of light-emitting diode (LED) technology represents a promising approach to improve plant growth and metabolic activities. The aim of this study was to investigate the effect of different light spectra: red (656 nm), blue (450 nm), red/blue (3:1), and white (peak at 449 nm) on biochemical properties, photosynthesis and gene expression in two lettuce cultivars (Lollo Rossa and Lollo Bionda) grown under different methods of nutrient solution replacement in hydroponics. Complete replacement and EC-based replacement of nutrient solution increased content of proline and soluble sugars and activity of antioxidant enzymes (CAT, GPX and SOD) under the red/blue LED and red LED light treatments in both cultivars. In addition, the red/blue and the monochromatic red light increased the soluble protein content and the antioxidant activity in the Lollo Rosa cultivar under the replacement method according to the needs of the plant. An increase in flavonoid content in the EC-based method in the Lollo Rosa variety treated with a combination of red and blue light was also observed. The red/blue light had the greatest induction effect on anthocyanin content, expression of the UFGT, CHS, and Rubisco small subunit genes, and the net photosynthetic rate. Data presented here will directly contribute to the development of nutrient solution and LED spectrum management strategies to significantly improve plant growth and metabolism, while avoiding water and nutrient waste, and environmental pollution.