Field experiment was conducted to explore the effects of different ratios of biogas slurry substitute chemical nitrogen fertilizers on yield, nutrient accumulation and fresh quality of lettuce (L. sativa). The results showed that the application of fertilizers and slurry could promote the growth of lettuce. The yields of treatments with 50%, 75% and 100% ratios of slurry were 9.1%, 16.1% and 10.3% respectively higher than that of 100% urea treatment, but chemical fertilizer as the basal got a better effect than slurry while biogas slurry got better growth and higher nutrient absorption as the after manuring compared to chemical N fertilizer. The economic yield increased with the increasing ratio of slurry. Compared to 100% chemical treatment, nitrate contents under biogas slurry application ratio of 50%, 75% and 100% were decreased by 13.6%, 14.3% and 11.3%, respectively. However, soluble sugar, Vc contents and free amino acid concentrations of 100% biogas slurry application were lower but were highest of 75% biogas slurry application. In conclusion, the proportion of 75% slurry application was the best fertilizer ratio.