A novel method to detect beta-antagonists in rat blood was developed by combining heart-cutting 2-D CE in a single capillary with on-column electrochemical (EC) detection. The blood sample was purified in first dimension by non-micellar CZE separation. Then only a desirable fraction of the first dimension separation was transferred into the second dimension of the capillary by pressure. Finally, the purified sample was separated by MEKC. On-line dual concentration method, field amplified sample stacking and sweeping, was employed to accumulate the purified components that effectively counteracted the sample zone diffusion during the mobilization pressure step. A field decoupler joint and an EC detection cell were employed for on-column EC detection. After optimizing the analytical conditions, the RSD of peak height, peak area and migration time were 2.1-4.3%, 1.5-3.9% and 0.7-1.8% (n=10), respectively. This method has been successfully applied to determine beta-antagonists in postdosing rat blood sample. The pharmacokinetic study also demonstrated that the proposed heart-cutting 2-D CE with on-column EC detection was convenient, sensitive and would become an attractively alternative method for on-line sample cleanup and separation in complex samples.