Abstract: The objective of the present study was to understand the effect of substitution chemical fertilizers with different proportions of bioorganic fertilizers on the soil physicochemical properties and the yield and quality of tea in Yunnan tea plantation, and the optimal substitution proportions would be proposed based on the study. In this study, chemical fertilization(CK1) and organic fertilizer substituting 30% of chemical nitrogen(N) fertilizer(CK2) were used as controls. Four substitution ratios of bio-organic fertilizer(bioorganic fertilizer replacing 30%, 40%, 50% and 60% of chemical N fertilizer respectively) were set. The effects of replacing chemical N fertilizer with bio-organic fertilizer on soil physicochemical properties, tea yield, and quality were analyzed. The results showed that, compared with CK1 and CK2, the substitution of bio-organic fertilizer for N fertilizer at different ratios increased soil available N by 10.9%–21.3%, available phosphorus(P) by 2.1%–10.1%, and available potassium by 11.2%–24.2%. Meanwhile, the N and P content in tea shoots were increased by 3.6%–8.9% and 7.6%–48.8%, and the amount of P uptake by 30.6%–78.8%. Although bio-organic fertilizer replacing chemical N fertilizer at different ratios had no effect on total tea yield, water extract, or caffeine content, compared with CK1 and CK2, the substitution of 30%–40% of N fertilizer with bio-organic fertilizer increased free amino acid and tea polyphenol contents by 12.9%–36.8% and 14.2%–83.5% in tea leaves, respectively. Comprehensive analysis of tea yield, quality, soil nutrients, and tea nutrient content indicated that replacing 30%–40% of N fertilizer with bio-organic fertilizer effectively improved soil nutrient availability and significantly enhanced tea quality. Therefore, it is recommended to adopt a substitution ratio of 30%–40% bio-organic fertilizer for N fertilizer in practical production to achieve sustainable soil management and optimize tea quality.