The detection of microRNA (miRNA), a gene-regulating factor, is crucial for predicting plant growth and development. In this study, we successfully developed a fluorescent nanosensor using nanomaterials and molecular beacons for in situ miRNA detection in plant cells. The molecular beacons were loaded onto 15 nm gold nanoparticles, effectively protecting them from DNase I degradation. The nanosensor exhibits remarkable specificity and detection capability with a detection limit of 1.0 nM, while maintaining stability in plant nutrient solutions. It is readily absorbed and transported by plant through both the symplasmic pathway and apoplastic pathway. Furthermore, it efficiently converts miRNA signals into fluorescent signals, providing valuable insights into the spatial and temporal distribution of miRNA in plants. Although to achieve more accurate detection, the detection limit of the designed nanosensor still needs to be further improved, but this technique offers a convenient and efficient approach, serving as a powerful tool for studying the mechanisms and regulation in plants.