There is a need to develop simple yet effective preconcentration methods to enhance concentration sensitivity for CE analysis of trace level analytes in real samples, particularly when commonly available but less sensitive detection methods, e.g., UV detection, are used. In this report, a hyphenated online preconcentration strategy combining dynamic pH junction with sweeping (i.e., dynamic pH junction-sweeping) was employed for the analysis of four toxic pyrrolizidine alkaloids (PAs) of senkirkine, senecionine, retrorsine, and seneciphylline in Chinese herbal medicine (Kuan donghua). Direct electrokinetically focusing of a large sample volume injection (up to 20% of capillary length) on the capillary was performed using the dynamic pH junction-sweeping method. A sample matrix consisting of 10 mm phosphate with 20% methanol at pH 4.0 and a BGE containing 20 mm borate, 30 mm SDS, and 20% methanol at pH 9.1 were utilized to realize dynamic pH junction-sweeping for PAs. This online preconcentration strategy resulted in sensitivity enhancement factors ranging from 23.8 to 90.0-fold for the four toxic PAs, giving an LOD as low as 30 ppb for the PAs. Critical factors such as sample matrix type, pH, and salt concentration were also examined to achieve higher sensitivity enhancement, shorter analysis time, and better resolution. The results indicate that the proposed dynamic pH junction-sweeping technique is a powerful alternative approach for identification and determination of trace levels of these toxic PAs and other hydrophobic, protonatable compounds in real samples.