The effects of gossypol residue on urea hydrolysis and ammonia volatilization in soil were explored by the methodof mixed culture with gossypolresidue，urea andsoil，and measuring the amount of residual urea and ammonia absorbed. The results showed that the residual urea increased and the detected ammonia decreased with the increase of gossypolresidue. Compared with thecontrol，the urea residue difference rate reached78.81%，and the inhibition rate of ammonia volatilization reached 80.40%. Changes in soil water content had a great impact on the activity of urease and urease-producingmicroorganisms，and the inhibitory effect of gossypol residue on urea hydrolysis and ammonia volatilization was more obviouswhen the activity was high. As the culture temperature increased，urea hydrolysis increased and ammonia volatilizationincreased rapidly. Gossypol residue had the best inhibitory effect on urea hydrolysis at35℃，the urea residue difference ratereached 79.72%. Gossypol residue had the best inhibitory effect on ammonia volatilization at 30℃，and the inhibition rate reached 55.34%. These results indicated that the gossypol residue has strong inhibitory effect on urea hydrolysis and ammoniavolatilization，and has potential for application.