Sugarcane is a prominent source of sugar and ethanol production. Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome, long breeding cycle, and recalcitrance to genetic transformation. The protoplast-based transient transformation system is a versatile and convenient tool for in vivo functional gene analysis;however, quick and effective transformation systems are still lacking for sugarcane. Here, we developed an efficient protoplast-based transformation system by optimizing conditions of protoplasts isolation and PEG-mediated transformation in S. spontaneum. The yield of viable protoplasts was approximately 1.26 × 107 per gram of leaf material, and the transformation efficiency of 80.19％could be achieved under the optimized condition. Furthermore, using this approach, the nuclear localization of an ABI5-like bZIPs transcription factor was validated, and the promoter activity of several putative DNase I hypersensitive sites (DHSs) was assessed. The results indicated this system can be conveniently applied to protein subcellular localization and promoter activity assays. A highly efficient S. spontaneum mesophyll cell protoplast isolation and transient transformation method was developed, and it shall be suitable for in vivo functional gene analysis in sugarcane.