We developed an enzyme-linked immunosorbent assay for an endocrine disrupter, nonylphenol, using a microreactor composed of two reaction vessels stacked vertically through a microfluid filter. The filters constructed by deep X-ray lithography possessed 2100 through-bores (phi 40 x 200 mu m) in polymethylmethacrylate sheets (phi 3 mm), which are appropriate for biochemical reactions. Through the optimization of the immunoassay, nonylphenol was quantitatively detected at the range of 0.1-10 ng/ml.