Next-generation sequencing has been widely applied to understand the complexity of non-coding RNAs (ncRNAs) in a cost-effective way. In this study, we developed mirTools 2.0, an updated version of mirTools 1.0, which includes the following new features. (1) From miRNA discovery in mirTools 1.0, mirTools 2.0 allows users to detect and profile various types of ncRNAs, such as miRNA, tRNA, snRNA, snoRNA, rRNA, and piRNA. (2) From miRNA profiling in mirTools 1.0, mirTools 2.0 allows users to identify miRNA-targeted genes and performs detailed functional annotation of miRNA targets, including Gene Ontology, KEGG pathway and protein-protein interaction. (3) From comparison of two samples for differentially expressed miRNAs in mirTools 1.0, mirTools 2.0 allows users to detect differentially expressed ncRNAs between two experimental groups or among multiple samples. (4) Other significant improvements include strategies used to detect novel miRNAs and piRNAs, more taxonomy categories to discover more known miRNAs and a stand-alone version of mirTools 2.0. In conclusion, we believe that mirTools 2.0 (18.104.22.168/mr2_dev and centre.bioinformatics.zj.cn/mr2_dev) will provide researchers with more detailed insight into small RNA transcriptomes.