Though the common vampire bat, Desmodus rotundus, is known as the main rabies virus reservoir in Latin America, no tools are available to investigate its antiviral innate immune system. To characterize the IFN-I pathway, we established an immortalized cell line from a D. rotundus fetal lung named FLuDero. Then we molecularly characterized some of the Toll-like receptors (TLR3, 7, 8 and 9), the three RIG-I-like receptor members, as well as IFNα1 and IFNβ. Challenging the FLuDero cell line with poly (I:C) resulted in an up-regulation of both IFNα1 and IFNβ and the induction of expression of the different pattern recognition receptors characterized. These findings provide evidence of the intact dsRNA recognition machinery and the IFN-I signaling pathway in our cellular model. Herein, we generated a sum of insightful specific molecular and cellular tools that will serve as a useful model to study virus–host interactions of the common vampire bat.