Discriminating self from nonself is fundamental to immunity. Yet, it remains largely elusive how the mechanisms of self and nonself discrimination originated. Sensing double-stranded RNA as nonself, the 2',5'-oligoadenylate synthetase (OAS)-ribonuclease L (RNase L) pathway represents a crucial component of innate immunity. Here, we combine phylogenomic and functional analyses to show that the functional OAS-RNase L pathway likely originated through tinkering with preexisting proteins before the rise of jawed vertebrates during or before the Silurian period (444 to 419 Mya). Multiple concerted losses of OAS and RNase L occurred during the evolution of jawed vertebrates, further supporting the ancient coupling between OAS and RNase L. Moreover, both OAS and RNase L genes evolved under episodic positive selection across jawed vertebrates, suggesting a long-running evolutionary arms race between the OAS-RNase L pathway and microbes. Our findings illuminate how an innate immune pathway originated via molecular tinkering.