Abstract: Leaves of Alamo switchgrass (Panicum virgatum) were used as materials to establish a transient expression system for mesophyll protoplasts cells. To determine the optimal conditions to obtain protoplasts from switchgrass, we designed a gradient experiment, with three replicates per treatment. First, we determined the optimal age of plants as the source of material. The leaves of etiolated seedlings grown for 1, 2, 3, and 4 weeks were subjected to enzymolysis for 8 h in a solution containing D-mannitol at 0.6 mol/L. Next, we determined the optimal osmotic pressure by exposing leaf material from 2-week-old etiolated seedlings to solutions containing D-mannitol at 0.4 mol/L, 0.5 mol/L, 0.6 mol/L, 0.7 mol/L and 0.8 mol/L. The optimal duration of enzymolysis was determined by subjecting leaf material from 2-week-old etiolated seedlings to enzymolysis for 6, 7, 8, 9, and 10 h in a solution containing 0.6 mol/L D-mannitol. This series of assays showed that the optimal conditions to isolate protoplasts were as follows: 2-week-old etiolated seedlings as the source material, and enzymatic hydrolysis in a solution containing 0.6 mol/L D-mannitol in the dark for 8 h at 28 ℃. The protoplasts obtained using this method were transformed with the plasmid LN-OsMYB103 by a polyethylene glycol-mediated method, and strong green fluorescence was detected by laser confocal microscopy. These results indicated that the protoplasts retained normal biological functions. In summary, we established an integrated transient expression system for protoplasts obtained from switchgrass leaves. This method will be very useful for functional genomics research on switchgrass.