Modification of nucleotides significantly increases the diversity of functional nucleic acids. As one of the most common modifications of RNAs， methylation of the 2'-hydroxyl-group of ribonucleotides (2'-O-methylation) has been found in various RNAs in eukaryotes. However， due to lack of efficient method for quantifying small RNA 3' terminal 2'-O-methylation， it is difficult to monitor the dynamic change of 3' terminal 2'-O-methylation during various biological process. Capitalizing on the finding that 3' terminal RNA 2'-O-methylation can inhibit the activity of poly(A) polymerase， an enzyme used in real-time quantitative polymerase chain reaction (RT-qPCR) assay， here we develop a poly(A)-tailed RT-qPCR method by which the 2'-O-methylation level of small RNAs such as microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs) can be directly quantified. With this method， we successfully determine the 2'-O-methylation level of miRNAs in Arabidopsis thaliana and mouse lung tissue， piRNA in human seminal plasma， and monitor the alteration of miRNA 2'-O-methylation in Drosophila Schneider 2 cells after knockdown of Drosophila methyltransferase protein Hua enhancer 1 (DmHen-1).