Substitution of chemical fertilizers with organic amendments recycle organic wastes and minimizes adverse environmental effects of mineral-fertilizer overuse. In China, cropping systems which utilize two rice plantings per year are the most important planting pattern for ensuring food security. To date, few studies have comprehensively evaluated the responses of soil fertility and rice performance in double rice cropping systems to the partial substitution of chemical nitrogen (N) with organic N under the same input rates of N, P and K. In addition, the influences of the mineral-to-organic substitution ratio and season on these responses remain poorly understood. Here, a 34-year field fertilizer trial was sampled to determine the impacts of organic substitution regime and growing season on soil biochemical properties and rice yield. Compared with the mineral NPK fertilizer regime, the replacement of 30 %, 50 % and 70 % of total N with organic N increased the yield of early rice by 4.5 %–8.3 %, respectively and that of late rice by 6.7 %–9.6 %. Moreover, the organic substitution regimes significantly improved soil biochemical indicators. The higher organic N substitution percentage resulted in higher values of many soil properties with the exception of TC:TN, fungi:bacteria and G⁺:G⁻, which tended to decrease with increasing substitution. Both fertilizer regime and growing season affected soil nutrient contents, enzyme activity and microbial community structure. The relative abundances of identified PLFA biomarkers 16:1ω5c, 16:1ω7c and 18:1ω7c were markedly higher (p ≤ 0.05) in the organic alternative treated soils than the NPK-treated soils in both seasons. Our results suggest that the substitution of 70 % chemical N with organic N is the most effective fertilizer practice for improving soil fertility and rice yield in double rice cropping systems.