Manipulating crop production and aquaculture in agro-ecosystems by utilizing the positive species interactions can not only increase the yield of crops, but also reduce the utilization of chemical fertilizers and pesticides. In this study, soil nematodes were used as a biological indicator to investigate the effects of co-culture of rice (Oryza sativa) and an exotic species Procambarus clarkii, aiming to evaluating the effect of rice-crayfish co-culture on soil health of agro-ecosystem. Soil samples were collected from rice monoculture and rice-crayfish co-culture at the tillering stage, heading stage and maturing stage of rice and the community structure of soil nematodes were investigated. The results showed that compared with rice monoculture, the percentage of bacterial-feeding nematodes increased by 26.52%, 19.85% and 32.65%, while the percentage of cp1 nematodes increased by 31.13%, 24.91% and 19.49% at the tillering, heading and maturing stages under rice-crayfish co-culture, respectively, indicating a faster nutrient cycling and higher nutrient level in rice-crayfish co-culture ecosystem. However, the percentage of cp3-5 nematodes and the maturity index were significantly lower in rice-crayfish co-culture than those in rice monoculture (P<0.05), indicating that the P. clarkii in co-culture may induce a simplified soil food web and a decreased soil biological stability, thus, further study is needed to monitor soil health in rice-crayfish co-culture ecosystem.