Abstract: To verify effect of biogas slurry application on growth and development environment of substrate cultivated pepper, a single variable method was used to study differences in changes in pepper quality, soil physicochemical properties, and rhizosphere microorganisms among five treatments: conventional cultivation（CK）, soil cultivation group（T₁）with biogas slurry application, soil cultivation group（T₂）with nutrient solution application, substrate cultivation group（T₃）with biogas slurry application, and substrate cultivation group（T₄）with nutrient solution application．Results showed that application of biogas slurry effectively improved quality of peppers, especially with a significant 53.3% increase in Vc content in the T₃ group compared to the CK group; improved soil physical and chemical properties; significantly increase relative abundance of actinomycetes, ascomycetes, etc．In addition, it was found that effect of applying biogas slurry in substrate cultivation was significantly better than that in soil．Applying biogas slurry in substrate cultivation can not only improve quality of peppers, but also improve basic physicochemical properties of substrate, change microbial community structure of rhizosphere substrate of peppers, and promote absorption and utilization of nutrients by crops．