In this study, the application of nonthermal plasma technology in the inactivation of ribonuclease was discussed, and the influence of acting gas and plasma generator on the inactivation performance was analyzed. The experimental results show that ribonuclease can be inactivated effectively by nonthermal plasma. Moreover, the inactivation performance will be obviously improved by mixing oxygen or water into the acting gas. Also, compared with surface dielectric barrier discharge plasma, the inactivation performance of ribonuclease is better by using floating-electrode dielectric barrier discharge generator.