Isolation of high-quality RNA free of contaminants, such as polyphenols, proteins, plant secondary metabolites, and genomic DNA from plant tissues, is usually a challenging but crucial step for molecular analysis. We developed a novel protocol based on the cetyltrimethylammonium bromide method to isolate high-quality RNA from blackberry plant tissues, especially fruits. Most DNA was removed when acetic acid was utilized, before RNA precipitation. Thus, lithium chloride, a reagent widely used for RNA purification, was not needed. The isolation time was shortened to less than 3 h.