In order to improve the utilization rate of fertilizer and cultivate high-quality muskmelon，the seeds of ‘Yangjiaomi’ melon were used as experimental materials in this study. Using peat∶vermiculite∶perlite = 1∶ 1∶1(volume ratio)as culture substrate，6%(T1)，12%(T2)，18%(T3) and 24%(T4) of bio-organic fertilizer were added into the substrate，respectively，and no bio-organic fertilizer added was used as CK. The effects of different addition levels of bio-organic fertilizer on physical and chemical properties of substrate，nutrient contents，substrate enzyme activities，and the growth，leaf photosynthetic parameters，fruit quality and yield of muskmelon were studied. The results showed that:(1)Adding a certain amount of bio-organic fertilizer significantly improved the ventilation and water holding status of the substrate，increased the pH value，EC value and nutrient contents，and increased the number of bacteria and fungi in the substrate.(2)Adding a certain amount of bio-organic fertilizer significantly improved the enzyme activities of the substrate. Under the T2 treatment，the activities of sucrase，urease，alkaline phosphatase and catalase in the substrate increased by 23.3%，25.2%，26.6% and 8.7%，respectively，compared with CK.(3)Adding appropriate amount of bio-organic fertilizer promoted the growth and photosynthetic rate of muskmelon plants. Under T2 treatment，the plant height，stem diameter，leaf area and root activity of melon were the highest，which were 21.6%，10.1%，20.6% and 21.3% higher than CK，respectively. The total chlorophyll content and net photosynthetic rate of melon leaves were 25.3% and 18.5% higher than CK，respectively.(4)Adding a certain amount of bio-organic fertilizer significantly improved the fruit quality and yield of melon，the contents of sucrose，glucose，total soluble sugar，soluble solids and soluble protein in muskmelon fruit under T2 treatment were significantly higher than those in other treatments，and the single fruit weight and yield per plant were increased by 23.5% and 38.6%，respectively，compared with CK. In summary，the addition of a certain amount of bio-organic fertilizer could improve the physical and chemical conditions of the substrate，increase the enzyme activities of the substrate，promote the growth of muskmelon plants，and improve the quality and yield of muskmelon fruits. The best treatment was T2，which was added with 12% bio-organic fertilizer.