Excessive application of chemical fertilizers leads to numerous problems such as soil acidification and compaction. Soybeans, as soil-improving crops, play a significant role in soil improvement. Their yield formation depends on the accumulation of dry matter in the later stage, and the slow-release property of organic fertilizers can meet this requirement. Current research focuses on the combined application mode of chemical and organic fertilizers. This study explored the impact of partial substitution of chemical fertilizers with organic fertilizers on soybean yield and soil fertility, which was of great significance. This experiment used ‘Wandou 37’ as the material and set different ratios of organic fertilizer substitution for chemical fertilizers to analyze the growth traits, yield components, nutrient absorption, and changes in soil physical and chemical properties of soybeans, and to screen the best substitution ratio, providing a theoretical basis for green soybean production. Suitable plots were selected in Mengcheng, Anhui Province, and six treatments were set up, including single application of chemical fertilizers (T₁) and five treatments of organic fertilizer substitution for 20% to 100% of chemical fertilizers (T₂ to T₆). Samples were collected at maturity to analyze relevant indicators. The results showed that the plant height, stem diameter, and dry matter weight of each treatment with organic fertilizer substitution were all improved to varying degrees compared with the single application of chemical fertilizers. The yield of each treatment increased first and then decreased with the substitution ratio. Among them, the treatment with 60% substitution of chemical fertilizers (T₄) had the largest increase in yield (29.33%), and the number of pods per plant, the number of seeds per plant, and the 100-seed weight all reached the highest. After applying organic fertilizers to substitute for chemical fertilizers, the contents of soil organic matter, total nitrogen, available phosphorus, and available potassium were significantly increased, and the microbial biomass carbon, nitrogen, and phosphorus also increased. Organic fertilizer substitution could increase soil pH, improve soil buffering capacity, and alleviate acidification in the long term. Partial substitution of chemical fertilizers with organic fertilizers can significantly promote soybean growth, increase yield, and improve soil quality. Among them, the comprehensive effect of 60% substitution ratio is the best, which has the benefits of increasing yield and sustainable soil improvement, and is the recommended optimal fertilization mode.