Epigenetic modifications of RNA play a critical role in RNA structure and gene regulation. Ribose 2’-O-methylation (Nm) is one of the most common modifications among them, featured with one methyl group added to the 2’ hydroxyl of the ribose moiety of RNA nucleosides. Although 2’-O-methylation was widely present in different type active RNAs, such as mRNAs, tRNAs, rRNAs, and even miRNAs, its biological functions and significance are still largely unknown due to the lack of accuracy and efficient identification screening tools. In this study, we first time established an enzyme-based high throughput Nm sites detection method with base resolution, which was named NJU-seq (Nm site Judge Universally Sequencing). We effectively identified Nm sites in human cultured cell lines, plants and animals with this new sequencing screening technique. Through this approach, we observed high abundance and complexity of Nm sties in cellular RNAs cross different species samples. Our study suggested that the function and regulation of Nm sites are more complicated than previous studies revealed and worth further mechanistic investigation.