The ubiquitin specific peptidase (USP) family is involved in many life processes, of which antiviral is also an important basic function. One of the more important ways is to activate interferon. In this study, we reported the antiviral function of the ubiquitin specific peptidase 5(USP5) gene in zebrafish. Evolutionary and comparative protein sequence analysis of the USP5 was performed. The localization of USP5 in FHM cells cytoplasm was determined. Overexpression of USP5 significantly evoked higher expression of mRNA that encode IFNφ1 and ISGs, the promoteractivities of IFNφ1 and IFNstimulated response element (ISRE) were augmented likewise. USP5 was also able to enhance the expression of RIG-I and activate higher levels of IFNφ1 stimulated by Poly (I: C). Viral infection and interference tests demonstrated that USP5 inhibited the replication of SVCV in vitro. In summary, this study reveals that USP5 is able to activate higher levels of interferon by increasing RIG-I protein levels, and thus implement antivirus functions.