A pressurized capillary electrochromatography (pCEC) method combined with an online concentration was developed for the separation and determination of five beta(2)-blockers (terbutaline, salbutamol, formoterol, procaterol and salmeterol) in human urine. A stearyl methacrylate-based monolithic column was prepared as the separation column. The various separation parameters including acetontrile concentration, applied voltage, pH and concentration of the running buffer were investigated. On-line concentration methods in combination of the chromatographic zone-sharpening effect and field-enhanced sample-stacking effect were utilized to improve detection sensitivity. The on-line concentration parameters including injection voltage, injection time, as well as sample matrix were systematically studied. Compared with the conventional sample injection, the on-line concentration technique appeared to improve the corresponding sensitivities 20 similar to 50-fold. Furthermore, good precision was obtained with relative standard deviations (RSDs) for retention times within 1.09% and for peak areas less than 3.55% (n = 5). The established method was successfully applied to the determination of above-mentioned beta(2)-agonists in urine samples. The recoveries of spiked urine samples were between 85.0 and 113.3% with RSDs less than 5.39%.