To clone Type I interferon (IFN-1) from epithelioma papulosum cyprini cells (EPC) of Pimephales promelas, the ifn-1 gene was amplified from EPC by RT-PCR, and the recombinant plasmid of pET-32a-IFN-1 was constructed and transformed into the host strain Transetta for expression. Sequence analysis showed that the coding region of ifn-1 gene was 552 bp in length and encoded 184 amino acids, which was closely related to grass carp interferon 1(CiIFN1). Recombinant soluble IFN-1 (rIFN-1) was induced in Transetta by IPTG and obtained by Ni-affinity purification. Then, anti-rIFN-1 polyclonal antibody was prepared by immunizing New Zealand white rabbits with rIFN-1 and could be used to detect endogenous IFN-1 in EPC cells. Quantitative PCR showed that the antiviral protein Mx1 was induced and spring viremia of carp virus (SVCV) replication as well as the production of cytopathic effect (CPE) was significantly inhibited when EPC was pre-incubated with rIFN-1, which indicated that rIFN-1 possessed antiviral activity in EPC cells.