The binding affinity of 17 beta-estradiol with an immobilized DNA aptamer was measured using capillary electrophoresis. Estradiol captured by the immobilized DNA was injected into the separation capillary using pH-mediated sample stacking. Stacked 17 beta-estradiol was then separated using micellar electrokinetic capillary chromatography and detected with UV-visible absorbance. Standard addition was used to quantify the concentration of estradiol bound to the aptamer. Following incubation with immobilized DNA, analysis of free and bound estradiol yielded a dissociation constant of 70 +/- 10 mu M. The method was also used to screen binding affinity of the aptamer for estrone and testosterone. This study demonstrates the effectiveness of capillary electrophoresis to assess the binding affinity of DNA aptamers.