The analytical method for calcium species in human blood was studied using ultrasonic-assisted dialysis coupled with capillary electrophoreisi (CE) inductively coupled plasma optical emission spectrometry (ICP-OES). The optimal ultrasonic dialysis conditions were 72 W power, 60 min extraction time and 70 μL volume of blood sample. Ultrasonic-assisted dialysis improves significantly the dialysis efficiecy by comparing with balance dialysis. The optimized conditions of separation and detection (a 120 cm length×100 μm i.d. fused-silica capillary, 20 kV separation voltage, 30 mmol/L Tris-HCl with pH7.4 buffer solution) for calcium species were achieved by CE-ICP-OES. The forms of calcium in the dialysate of human blood have five different species. The concentration of free Ca2+ in the dialysate was 0.7 mg/L. The hyphenated technique is simple and convenience, which is fit for the separation and analysis of metal-containing biological samples.