A plant protoplast system can be used to study transient gene expression, protein subcellular localization, protein-protein interaction and protein activity as well as gene function. The application of heterologous protoplast systems to the expressed proteins may exhibit aberrant trait. To avoid this mistaking, it is necessary to establish and apply the host protoplast systems. In wheat, the PEG-mediated gene protoplast transformation is hampered by the release of nucleases from the protoplasts, which leads to extensive degradation of plasmid DNAs. In this study, in order to get the high efficiency of the wheat protoplast transformation, means including inhibiting the nucleases activity and enlarging the plasmid quantity were used. The results showed that the protoplast transformation efficiency could be improved through double-fold usage of the plasmid quantity and inhibiting the nucleases activity by low transformation temperate. Consequently, the wheat protoplast transformation efficiency was raised to 85 %. Moreover, this wheat protoplast transformation system was used to study the expression protein subcellular localization of 2 wheat disease-related genes. This research has reference value for the future relevant work