Objective It is important to develop an efficient protoplast isolation and transient expression system in high-quality forage grass， Pennisetum sinese Roxb.Method In this study， the pCAMBIA1304-LbCas12a-PsC3H53cr vector was constructed through the CRISPR-Cpf1 system. The plasmid was mixed with protoplast solution and transformation solution. The protoplasts were cultured under weak light conditions. Four treatments（1.5%， 2.0%， 2.5%， 3.0%） and seven sets of enzyme digest times （3， 4， 5， 6， 7， 8， 9 h） were set for the enzymatic hydrolysis solution of cellulase R-10. The enzymatic hydrolysis conditions suitable for P. sinese were optimized to improve its yield and activity. Finally， polyethylene glycol（PEG） was used to mediate protoplast transformation.Result The enzyme digestion for 7 h was optimal with the combination of 2.5% cellulase， 0.5% pectinase and 0.4% dissociative enzyme， and the yield of protoplasts was up to 1.712 × 10⁶ cells/（g·FW）. PEG method can successfully mediate plasmid transformation into protoplasts of P. sinese and green fluorescence protein could be observed under fluorescence microscope.Conclusion A transient expression system and genetic transformation system were successfully established in P. sinese. After optimizing the best enzymatic hydrolysis combination and time， the transient transformation of P. sinese bioplastids could be achieved through PEG mediation， laying the foundation for the subsequent regeneration and variety breeding of P. sinese.