Eriocheir sinensis and Procambarus clarkii are two important economic aquatic products in China, the development of the two ecological polyculture can not only make full use of the bait resources, but also promote the transformation from traditional culture model to modern culture model. However, in actual production, the attacking and fighting behavior caused by problems such as breeding density, stocking size, sex ratio and other issues greatly reduce the survival rate of E. sinensis and P. clarkii. Thus, in order to explore the key factors influencing the aggressive and fighting behaviors of E. sinensis and P. clarkii under different polyculture conditions, several indoor behavioral observation experiments were carried out to analyze the effects of size, density, gender on the fighting behavior between E. sinensis and P. clarkii. The results showed that the number of attacks and fights between E. sinensis and P. clarkii increases as their size increases. When the size of E. sinensis and P. clarkii are different, the size of E. sinensis showed a significant role in the intensity of their fighting. The bigger E. sinensis get, they attacked and fought with each other more frequent. In polyculture mode, light had no significant effect on their fighting behavior. As the density of P. clarkii increased, the frequency and intensity of fights first increased and then decreased. There was a positive correlation between the content of lactic acid in hemolymph and cheliped muscle and the times of fighting The male of E. sinensis and P. clarkii are more aggressive, as male fights more than female at all density groups. In conclusion, in the practice of polyculture, the culture density and size of E. sinensis had a significant effect on fighting behavior, the traditional mode which releasing P. clarkii before E. sinensis can reduce the coexistence time of large E. sinensis and P. clarkii, thus mitigating the impact of fighting between E. sinensis and P. clarkii. Meanwhile, a proper gender proportion and feeding management can alleviate the cannibalism between E. sinensis and P. clarkii. This study can provide some theoretical guidance and reference for practical production.