Nitrogen reduction combined with biochar application is of great significance for improving land productivity, improving soil carbon sequestration and mitigating climate warming. Based on the field experiment, five nitrogen fertilizer dosage gradients (T0~T4) were set:The response of organic carbon mineralization and enzyme activity to nitrogen reduction combined with biochar application in paddy fields was revealed by using the principle of isonitrogenous fertilizer (100% N, 90% N, 80% N, 70% N, 60% N). The results showed that compared with T0 treatment, the soil total nitrogen, alkaline hydrolysable nitrogen and available phosphorus in T3 treatment (70% N +7.5 t/hm² biochar nitrogen) were significantly increased by 6.67%, 8.36% and 30.94% (P < 0.05), respectively. The content of available potassium in T4 treatment was the highest and significantly increased by 23.78% (P < 0.05). Soil organic carbon (SOC) content increased with the increase of nitrogen reduction combined with biochar application ratio. Compared with before mineralization, SOC, microbial biomass carbon (MBC) and microbial entropy (qMB) decreased by 1.39~1.75 g/kg, 24.62~67.57 mg/kg and 0.13%~0.32% (P < 0.05) after mineralization. SOC mineralization rate reached the peak value on the first day of culture, decreased rapidly in the first stage (1~6 days), decreased slowly in the second stage (6~30 days), and tended to be stable in the third stage (30~45 days). The relationship between mineralization rate and culture time was logarithmic (P < 0.01). At the end of culture, the SOC cumulative mineralization amount and cumulative mineralization rate varied from 1.39~1.75 g/kg and 6.02%~8.43%, respectively, and T3 treatment was the lowest. Compared with CK and T0 treatments, the activities of catalase, urease and sucrase in T3 treatment were the highest, and the activities of acid phosphatase in T1 treatment were the highest. The highest rice yield was found in T3 treatment (7.37 t/hm²), which was 39.58% higher than that in T0 treatment (P < 0.05). In conclusion, 30% reduction of nitrogen fertilizer combined with biochar can significantly improve soil fertility, reduce SOC mineralization, increase soil carbon sequestration, improve soil enzyme activity and rice yield.