DEAD-box helicase 41 (DDX41) is a key cytosolic DNA sensor playing critical roles in the regulation of type I IFN responses, and their functions have been well-characterized in mammals. However, little information is available regarding the function of fish DDX41. In this study, a DDX41 gene, named On-DDX41, was identified in Nile tilapia, Oreochromis niloticus. The predicted protein of On-DDX41 contains several structural features known in DDX41, including conserved DEADc and HELICc domains, and a conserved sequence "Asp-Glu-Ala-Asp (D-E-A-D)". On-DDX41 gene was constitutively expressed in all tissues examined, with the highest expression level observed in liver and muscle, and was inducible after poly(I:C) stimulation. Moreover, the overexpression of On-DDX41 can elicit a strong activation of both zebrafish IFN1 and IFN3 promoter in fish cells treated with poly (dA:dT). The present study thus contributes to a better understanding of the functional properties of DDX41 in fish.