Effective recognition and quantitative analysis of the prion protein are important in drug discovery and diagnosis for prion diseases, such as bovine spongiform encephalopathy and Creutzfeldt-Jakob diseases. We have developed a high-throughput method for a specific and sensitive determination of prion protein on a solid-phase membrane, based on a chemiluminescence reaction of aptamer with 3,4,5-trimethoxyphenylglyoxal. This method using aptamer is facile, inexpensive and convenient for the detection of the prion protein on a membrane, indicating a lower detection limit of ca. 4.2 pmol spot(-1).