To optimize the supravital staining of plant vacuome with neutral red, this study used root tips of wheat, rice and mung bean as materials and 1/3000 neutral red as the staining agent, and carried out conditional optimization experiments on material selection, preparation and preservation of staining solution, and staining duration. Results showed that wheat seeds required simple culture conditions and had a short growth period; the vacuoles in the root apical meristem of wheat were stained uniformly with high specificity, and the observation effect was better than those of rice and mung bean. Comparisons of different staining durations indicated that insufficient staining occurred at 10 min, while over-staining or altered cell morphology tended to appear at 20 min and above. Staining for 15 min yielded appropriate color development and intact vacuole structure, which was determined as the optimal duration. Under the optimized conditions of wheat as material, freshly prepared neutral red solution, and 15 min staining, distinct developmental morphological differences were clearly observed among the primary small vacuoles in the apical meristem, small vacuoles in the elongation zone, and the central large vacuole in the maturation zone. The optimized protocol for supravital staining of the plant vacuome established in this study was simple to operate and stable in observation effect, and provided a reference for relevant supravital staining experiments.