Abstract: In order to evaluate the antiviral activity against IHNV of rainbow trout IFN-γ2, the target gene (471 bp) was successfully amplified from the cultured primary head kidney leucocyte cultures which had been stimulated by PHA using the primers which were designed based on the NCBI reference sequence. Then, the gene was inserted into the pET32a vector and was expressed in E.coli Rosetta. The result of SDS-PAGE showed that the recombinant protein was successfully expressed in the inclusion bodies, which was about 38.4 ku. The recombinant protein could be simply purified by just washing two times. The antiviral ability of the refolding protein was evaluated in CHSE-214 cells. The activity of rtIFN-γ2 against IHNV was 6.63×10⁶ U/mg. Based on this, the rainbow trout were injected with rtIFN-γ2. The results of real-time PCR indicated that rtIFN-γ2 was potent to induce the comparable levels of IRF-1, IRF-2, IFN-I, IFN-γ and Mx transcription in head kidney, spleen and liver. Generally, the antiviral state on day 1 post immunization was stronger than that on day 2 post immunization. In addition, its protection against IHNV was 40% and 80% when the challenge was on day 1 and day 2 post immunization, which was closely correlated with the kinetic profile of the antiviral state. In conclusion, our study here showed that the rtIFN-γ2 of rainbow trout with the activity was successfully produced in the prokaryotic expression system. Furthermore, the rtIFN-γ2 could elicit the ideal antiviral state and provide the subsequent protection against IHNV.