Next-generation sequencing has been widely applied to understand the complexity of non-coding RNAs (ncRNAs) in the last decades. Here, we present CPSS 2.0, an updated version of CPSS 1.0 for small RNA sequencing data analysis, with the following improvements: (i) a substantial increase of supported species from 10 to 48, (ii) improved strategies applied to detect ncRNAs, (iii) more ncRNAs can be detected and profiled, such as lncRNA and circRNA, (iv) identification of differentially expressed ncRNAs among multiple samples, (v) enhanced visualization interface containing graphs and charts in detailed analysis results. The new version of CPSS is an efficient bioinformatics tool for users in non-coding RNA research.