Exoribonucleases are frequently used as nuclei acids detection tools for their sequences, modifications, and structures. Escherichia coli ribonuclease R (EcR) is the prototypical exoribonuclease of the RNase II/RNB family degrading RNA in the 3'-5' direction. Different from RNase II, EcR is capable of degrading structured RNA efficiently, which makes it a potential analysis tool for various RNA species. In this work, we examined the nuclease activity of EcR degrading a series of RNA substrates with various sequences. Our biochemical work reveals that EcR is significantly sensitive to cytosine compared with other bases when catalyzing RNA degradation. EcR shows higher cytosine sensitivity compared to its homolog RNase II when degrading RNAs, and the hydrolysis process of EcR is transiently halted and produces apparent intermediate product when the 1-nt upstream of C is A or U, or G. Furthermore, the substitution of glycine with proline (G273P) in EcR enhances its cytosine sensitivity. These findings expand our understanding of EcR enzymatic activities. The EcR G273P mutant bearing higher cytosine sensitivity could help enrich cytosine trails in RNAs and will have potential implications in the detection and analysis of various RNA species especially small RNAs in biological and clinical samples.