Single-molecule fluorescence in situ hybridization (smFISH) is a powerful method for the visualization and quantification of individual RNA molecules within intact cells. With its ability to probe gene expression at the single cell and single-molecule level, the technique offers valuable insights into cellular processes and cell-to-cell heterogeneity. Although widely used in the animal field, its use in plants has been limited. Here, we present an experimental smFISH workflow that allows researchers to overcome hybridization and imaging challenges in plants, including sample preparation, probe hybridization, and signal detection. Overall, this protocol holds great promise for unraveling the intricacies of gene expression regulation and RNA dynamics at the single-molecule level in whole plants.