<p>Nitrogen (N) availability is a key factor for sustainable production in rice-crayfish co-culture system (RC), and the appropriate N rate should be determined in RC by considering grain yield, N uptake, grain quality as well as soil fertility under different N rate.&#xa0;Between 2020 and 2022, different N levels (0, 60, 120, 180 and 240&#xa0;kg N ha^− 1) were tested to explore the optimal amount of fertilizer application in RC. Soil NO₃⁻-N, NH₄⁺- N and inorganic N (N_min) increased as N rate increased at the tillering stage (TS) and full heading stage (HS) of rice, and soil urease and catalase activity in N treatments increased significantly. The cumulated dry matters and plant N of N treatments were also increased by 4%~69% and 23%~164% (TS), 16%~69% and 30%~98% (HS), and 12%~49% and 26%~70% (mature stage), respectively. N increased rice yield by 8.47%~43.22%, the yield stability of N120 was highest, and the total benefit reached its peak under N120 and N180. The optimal N rate was 136 ~ 143&#xa0;kg N·ha^− 1, achieving maximum yield (7,861&#xa0;kg·ha^− 1) and maximum income (16,433 Yuan ha^− 1). N addition increased brown rice rate and milled rice rate by 4.95%~13.14% and 0.31%~10.87%, and reduced chalky rice rate by 19.57%~45.82%. After N fertilization, microbial biomass carbon, microbial biomass N and microbial biomass phosphorus were increased. <i>AOA</i>, <i>AOB</i>, <i>nir</i>S, <i>nir</i>K, <i>nos</i>Z, and <i>nar</i>G of 0–20&#xa0;cm soil were also increased.&#xa0;N fertilizer rates of RC systems should be 136 ~ 143&#xa0;kg N ha^− 1 to achieve a high grain yield with high soil microbial diversity and suitable water quality for crayfish. N addition had similar water quality than the control after 7 days of base fertilization but had higher soil N for rice that N addition is beneficial for both crayfish feeding and rice production.</p>