As a new wave of technology, polymethacrylate-based monolithic column was prepared and its application in the separation of three kinds of microcystins (MCs) in pressurized capillary electrochromatography with ultraviolet detection was studied. The key factors affecting the separation performance, such as monolithic column, pressure of the pump, component and concentration of mobile phase and the voltage, were investigated and optimized in detail. A baseline separation could be achieved in less than 6 min using a 5 mM borate buffer with a pH of 9.6 and 10% acetonitrile as the mobile phases in isocratic elution, under a voltage of +13 kV and a supplementary pressure of 7.5 M Pa. The calibration curves were linear with correlation coefficient r > 0.998 over a range of 0.10-25.00 mg/L. The LODs for the three MCs were in the range of 0.03-0.09 mg/L. This method was successfully applied to separate MCs from other compounds in spiked tap water after solid-phase extraction. The lower LODs for MC-LR, MC-YR and MC-RR were obtained to be 0.10, 0.13, 0.16 mu g/L, respectively. These results make it clear that this proposed system is accurate and robust enough to be used as a fast separation tool for routine monitoring of MCs in real water samples.