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The binding of estrogen receptor (ER) to estrogen response element (ERE) is essential for genomic pathways of estrogens and gel-based electrophoretic mobility shift assay (EMSA) is commonly used for analyzing ERE binding. Gel-based EMSA, however, requires the use of hazard radio isotopes and they are slow, labor-intensive and difficult to quantify. Here, we present quantitative affinity assays based on microchip electrophoresis using PEG-modified glass microchannels, which bear neutral surfaces against the adsorption of acidic DNA molecules and basic ER proteins. We first demonstrated the feasibility of the method by measuring binding constants of recombinant ERalpha and ERbeta with a consensus ERE sequence (cERE, 5'-GGTCAGAGTGACC-3') as well as with an ERE-like sequence (ERE 1576, 5'-GACCGGTCAGCGGACTCAC-3'). Changes in mobility as a function of protein-DNA molar ratios were plotted and the dissociation constants were determined based on non-linear curve fitting. The minimum amount of ER proteins required for one assay was around 0.2 ng and the run time for one chip analysis was less than 2 min. We further measured the estrogenic compound-mediated dissociation constants with recombinant ER proteins as well as with the extracted ERbeta from treated and untreated A549 bronchioloalveolar carcinoma cells. Dissociation constants determined by this method agree with the fact that agonist compounds such as 17beta-estradiol (1.70 nM), diethylstilbestrol (0.14 nM), and genistein (0.80 nM) assist ERE binding by decreasing the constants; while antagonist compounds such as testosterone (140.4 nM) and 4-hydroxytamoxifen (10.5 nM) suppress the binding by increasing the dissociation constant.