The cell wall, serving as the exoskeleton of plants, is naturally a barrier to resist external stresses. Protoplasts can be obtained by dissolving the cell walls of plant cells without damaging the cell membrane, and are widely used in the rapid propagation, transgenic breeding, and somatic hybridization of plants. However, to regenerate the cell wall is a precondition for cell division. Therefore, to study the culture condition and influencing factors during the cell wall regeneration of protoplasts is vital. Traditionally, culture medium is used to cultivate protoplasts, but it has some disadvantages. Herein, a microfluidic system with crossed channels was constructed to isolate and cultivate the protoplasts of tobacco. Then, the cell wall regeneration of the tobacco protoplasts was also studied based on this microfluidic system. It was found that, compared with the control, benzo-(1, 2, 3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) could accelerate the regeneration of the cell wall, while Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) could inhibit the regeneration of the cell wall within 24 h. To conclude, this study demonstrated that a crossed microfluidic chip could be an effective tool to study cell wall regeneration or other behavior of plant cells in situ with high resolution. In addition, this study revealed the rate of cell wall regeneration under BTH and Pst DC3000 treatment.