Ribonuclease I belongs to a class of nonspecific endoribonucleases and plays many important roles in a variety of biological and cellular processes. While their ubiquitous nature and high activity contribute to the well‐known problem of RNase contamination in experimentation, their abundance in bacteria can potentially be leveraged as a biosensor target. As a result, there is substantial interest in generating a specific and reliable probe for RNase detection for a variety of purposes. To that end, we report on our unintentional discovery of the RNase I probe RFA13‐1 isolated through in vitro selection with the crude extracellular mixture from
Clostridium difficilecontaminated with Klebsiella aerogenesas a selection target. Characterization of RFA13‐1 reveals that it exhibits high sensitivity to Escherichia coliRNase I with a detection limit of 1.39 p m. Furthermore, RFA13‐1 also shows high specificity for RNase I produced only in select bacteria from the Enterobacteriaceaefamily. As a result, this probe offers a simple tool for RNase I detection with potential applications in RNase functional studies, ribonuclease contamination monitoring, and bacterial detection.