The effects of nitrogen fertilizer application on nitrogen surplus and soil microbial community in paddy soil under the rice-crayfish co-culture system(RC)were explored，in order to ensure food security and reduce soil active nitrogen loss，and provide theoretical support for sustainable rice production. The field experiment used RC cultivation mode as the research object，with no nitrogen application as the control(CK)，and four nitrogen application rates(N60，N120，N180，N240)were set up to study the effects of different nitrogen application rates on nitrogen balance(the nitrogen balance between nitrogen input and nitrogen output)and soil microbial community characteristics under the RC. The proportion of nitrogen output to nitrogen input in the Procambarus clarkii system was 45.9%(2021)and 57.8%(2022)，respectively. The proportion of nitrogen output to nitrogen input in adult crayfish was 27.2% (2021)and 32.6%(2022)，respectively. A large amount of nitrogen was retained in the crayfish system. In the paddy field model，the apparent nitrogen balance of CK and N60 was negative，while the apparent nitrogen balance of N120，N180 and N240 was positive. Among them，N240 had the largest nitrogen surplus，reaching N 130.4 kg/hm²(2021)and N 163.1 kg/hm²(2022)，respectively. The increase in fertilizer nitrogen input was beneficial for the accumulation of ammonium nitrogen，nitrate nitrogen，and inorganic nitrogen in soil(0-60 cm)，which decreased with the increase of soil depth(0-60 cm)；the inorganic nitrogen content in different soil layers in 2022 was higher than that in 2021. Soil AOA，AOB，nirS，nirK，nosZ and narG(0-20 cm)all increased with the increase of fertilizer nitrogen input. The recommended suitable nitrogen fertilizer application amount was N 120-180 kg/hm². The apparent nitrogen balance in the rice field was positive，and the maximum nitrogen surplus was 103.5 kg/hm²，which met the recommended nitrogen surplus standards of the Netherlands MINAS and EU(N 100 and 80 kg/hm²). Long term rice-crayfish co-culture and increased application of nitrogen fertilizer were beneficial for increasing soil inorganic nitrogen content and expanding soil nitrogen pool.