Abstract: Interferon regulatory factor 3 （IRF3） regulates the expression of interferon （IFN）, and which mediates the host defense against pathogen infection. In order to obtain the recombinant protein of Trachinotus ovatus IRF3 （TroIRF3） and its polyclonal antibody, in the report, the pET32a（+）/Escherichia coli BL21（DE3） expression system was used for the inducible expression of TroIRF3. Under the conditions, 0.1 mmol·L⁻¹ IPTG, 24 h, 20 ℃, the soluble recombinant protein rTroIRF3 was obtained. The molecular mass of the rTroIRF3 protein was 51 kDa. The rTroIRF3 protein was purified by nickel affinity chromatography. The purified fusion protein rTroIRF3 was used as an antigen to immunize mice for the preparation of the polyclonal antibody. ELISA and western blotting were performed. The results indicated that the total titer of the polyclonal antibody of rTroIRF3 was 1∶25 600, the polyclonal antibody could bind specifically to the recombinant protein, and which can be used as a good experimental material for the subsequent mechanism analysis of TroIRF3 during pathogen infection.