Accumulating evidences suggest that microRNA (miRNA) is not a single sequence, but a cluster of isomiR sequences with various 5’/3’ ends and length distributions. The typical analysis always focuses on the annotated canonical miRNAs, although the multiple isomiRs have been reported as potential regulatory molecules with versatile biological roles. Herein, we develop an approach to analyze miRNA at the miRNA/isomiR levels. Firstly, isomiR expression profiles are estimated based on miRNA locus. Secondly, isomiR expression profiles are compared between clustered/homologous miRNAs. Thirdly, isomiR expression profiles are assessed at the isomiR level. Those isomiRs with the same “seed sequences” are classified as a special miRNA class. Finally, isomiR:mRNA interaction is predicted at the isomiR level, and target mRNAs and functional enrichment analysis can be performed to explore biological roles. Furthermore, the approach is applied to analyze miRNA at the miRNA/isomiR levels using small RNA sequencing datasets of tumor and normal cells. The analysis at the miRNA/isomiR levels will track miRNA maturation process and biological genesis, and provide more points to explore versatile biological roles of the small non-coding RNAs.