The concomitant cloning of RNA degradation products is a major concern in standard small RNA-sequencing practices. This not only complicates the characterization of bona fide sRNAs but also hampers cross-batch experimental replicability and sometimes even results in library construction failure. Given that all types of plant canonical small RNAs possess the 3' end 2'-O-methylation modification, a new small RNA sequencing (sRNA-seq) method, designated as PBOX-sRNA-seq, has been developed specifically to capture this modification. PBOX-sRNA-seq, as its name implies, relies on the sequential treatment of RNA samples with phenylboronic acid-polyacrylamide gel electrophoresis (PBA-PAGE) and sodium periodate (NaIO4) oxidation, before sRNA library construction and sequencing. PBOX-sRNA-seq outperformed separate treatments (i.e. PBA-PAGE only or NaIO4 only) in terms of the depletion of unmethylated RNA species and capture 2'-O-modified sRNAs with extra-high purity. Using PBOX-sRNA-seq, we discovered that nascent miRNA-5p/-3p duplexes may undergo mono-cytidylation/uridylation before 2'-O-methylation. We also identified two highly conserved types of 5'-tRNA fragments (tRF) bearing HEN1-independent 2'-O modification (mainly the 13-nt tRF-5aAla and the 26-nt tRF-5bGly). We believe that PBOX-sRNA-seq is powerful for both qualitative and quantitative analyses of sRNAs in plants and piRNAs in animals.