An advanced approach is developed in this work for simultaneous on-line separation and digestion of proteins by combining the Off-Gel isoelectric focusing (IEF) and enzymatic nanoreactor enhanced proteolysis. The nanoreactor was prepared by preloading trypsin in amino-functionalized macroporous silica, and then directly added into Off-Gel wells. With the nanoreactor loaded Off-Gel device, effective digestion of proteins happened during IEF electrophoresis to generate directly fractionated tryptic peptides, which not only accelerated the experimental flow but also avoided sample loss, leading to a more comprehensive protein identification from complex biological samples. A successful identification of 3592 proteins was achieved from Hela cell line by using the approach followed with LC-MS/MS analysis, while only 1877 proteins were identified from the same sample when using standard in-solution proteolysis followed with Off-Gel electrophoresis and then LC-MS/MS analysis. Therefore, we have demonstrated that the approach can greatly simplify high-throughput proteomics and significantly improve protein identification.