In this article, we developed a membrane-based enzyme micro-reactor by directly using commercial polystyrene-divinylbenzene cation-exchange membrane as the support for trypsin immobilization via electrostatic and hydrophobic interactions and successfully applied it for protein digestion. The construction of the reactor can be simply achieved by continuously pumping trypsin solution through the reactor for only 2 min, which was much faster than the other enzyme immobilization methods. In addition, the membrane reactor could be rapidly regenerated within 35 min, resulting in a "new" reactor for the digestion of every protein sample, completely eliminating the cross-interference of different protein samples. The amount and the activity of immobilized trypsin were measured, and the repeatability of the reactor was tested, with an RSD of 3.2% for the sequence coverage of cytochrome c in ten digestion replicates. An integrated platform for protein analysis, including online protein digestion and peptide separation and detection, was established by coupling the membrane enzyme reactor with liquid chromatography-quadrupole time-of-flight mass spectrometry. The performance of the platform was evaluated using cytochrome c, myoglobin, and bovine serum albumin, showing that even in the short digestion time of several seconds the obtained sequence coverages was comparable to or higher than that with in-solution digestion. The system was also successfully used for the analysis of proteins from yeast cell lysate.