The invention discloses recombination rainbow trout I type interferon, and a prokaryotic expression and purification method and application thereof. The prokaryotic expression and purification methodcomprises the steps of obtaining a rainbow trout I type interferon gene, screening the rainbow trout I type interferon gene, constructing a prokaryotic expression carrier, expressing and purifying therecombination protein and the like, so that the prepared rainbow trout I type interferon has favorable bioactivity, is safe and effective and can be used for preventing virus infection diseases for aquaculture, and has favorable application prospects.