Taking‘Yidalishengcai’as test material，adopting greenhouse cultivation in spring and summer 2 seasons，this paper studied the effects of different substrate thickness（6，10，14 cm）on root system environment，yield and quality of lettuce．The results showed that the thicker was the substrate，the less was the rangeability in temperature of lettuce root system and moisture content of substrate．The leaf area and leaf number treated with 10 cm and 14 cm thickness were superior than that treated by 6 cm thickness．And the single plant fresh weight，dry weight，soruble protein，soruble solid matter and VC content were all higher than that treated by 6 cm thickness．While，the nitrate content was remarkably lower than that treated by 6 cm thickness．Overall considering the cultivation frame load-bearing and cost input，10 cm thick seedling culture substrate mixed with sheep manure compound substrate（3 V∶2 V）was suitable to be used in lettuce layered cultivation．