Interferon plays role in innate immune responses through upregulation of costimulatory molecules and induction of proinflammatory cytokines. interferons including interferon alpha (IFNA). The present study characterized IFNA cDNA and predicted protein. The interferon’s play a great role in protection from infections, which have been called by microorganisms, and also have powerful antiproliferation and immunomodulation activity. The purposes of study: cloning and expression of horse leukocyte interferon and purification the product protein. The results and discussion : In the result we isolated (DNA) from equine leukocyte in blood, which was used in the quality of the matrix for amplification of α-interferon gene with PCR HELP, and isolation gene α-interferon and transformation in vector puc18 and expression vector PET24b (+) and recombinant plasmid was transformed into E. coli strain BL21( codon plus 440) induction with IPTG. The results showed the protein having the same molecular weight as horse interferon alpha about 5.81 kDa