On the basis of an europium (III)-doped Prussian blue analog film modifying platinum electrode as the working electrode, a Ru(bpy)₃²⁺-based electrochemiluminescence (ECL) assay coupled with capillary electrophoresis has been first established for the determination of ketotifen fumarate (KTF). Analytes were injected onto a separation capillary of 50 cm length (50 μm i.d., 360 μm o.d.) by electrokinetic injection for 10 s at 10 kV. Parameters related to the separation and detection were discussed and optimized. It was proved that 15 mM phosphate buffer at pH 8.0 could achieve the most favorable resolution, and the highest sensitivity of detection was obtained using the detection potential at 1.25 V and 5 mM Ru(bpy)₃²⁺ in 100 mM phosphate buffer at pH 8.0 in the detection reservoir. Under the optimized conditions, the ECL intensity was in proportion to KTF concentration over the range from 3.0 × 10⁻⁸ to 5.0 × 10⁻⁶ g mL⁻¹ with a detection limit of 2.1 × 10⁻⁸  g mL⁻¹ (3σ). The relative standard deviations of the ECL intensity and the migration time were 0.95 and 0.26%, respectively. The developed method was successfully applied to determine KTF contents in pharmaceuticals and human urine with recoveries between 99.5 and 107.0%.