The relevance of RNA in many biological functions has been recognized, broadening the scope of RNA research activities, from basic to applied sciences, also aiming the translation to clinical fields. The preparation and purification of RNA is a critical step for further application, since the quality of the template is crucial to ensure reproducibility and biological relevance. Therefore, the establishment of new tools that allows the isolation of pure RNA with high quality is of particular importance. New chromatographic strategies for RNA purification were considered, exploiting affinity interactions between amino acids and nucleic acids. In the present study, a single arginine-affinity chromatography step was employed for the purification of RNA from a total eukaryotic nucleic acid extract, thus eliminating several steps compared with current RNA isolation procedures. The application of this process resulted in a high RNA recovery yield of 96 ± 17% and the quality control analysis revealed a high integrity (28S:18S ratio = 1.96) in RNA preparations as well as a good purity, demonstrated by the scarce detection of proteins and the reduction on genomic DNA contamination to residual concentrations. Furthermore, the performance of the new RNA isolation method was tested regarding the applicability of the isolated RNA in modern molecular biology techniques. Hence, this new affinity approach will simplify the isolation and purification of RNA, which can bring great improvements in biomedical investigation.