Differential expression analysis using high-throughput RNA sequencing (RNA-seq) data is widely applied in transcriptomic studies and many software tools have been developed for this purpose. Active development of existing popular tools, together with emergence of new tools means that studies comparing the performance of differential expression analysis methods become rapidly out-of-date. In order to enable researchers to evaluate new and updated software in a timely manner, we developed DEAR-O, a user-friendly platform for performance evaluation of differential expression analysis based on RNA-seq data. The platform currently includes four of the most popular tools: DESeq, DESeq2, edgeR and Cuffdiff2. Based on the DEAR-O platform, researchers can evaluate the performance of different tools, or the same tool with different versions, with a customised number of biological replicates using already curated RNA-seq datasets. We also initiated an online forum for discussion of RNA-seq differential expression analysis. Through this forum, new useful tools and benchmarking datasets can be introduced. Our platform will be actively maintained to ensure new major versions of existing tools and new popular tools are included. DEAR-O will serve the community by providing timely evaluations of tools, versions and number of replicates for RNA-seq differential expression analysis.