A method using immunoaffinity as a purification step for the determination of aflatoxin M1in cheese is described. A simple solvent extraction with dichloromethane followed by a washing step with N‐hexane gives a prepurified extract. A comparison between two ways of aflatoxin M1purification, by solid‐phase extraction clean‐up and by immunoaffinity, was carried out. The use of immunoaffinity columns containing monoclonal antibodies against aflatoxin M1gives the best result, i.e. a very clean preparation containing purified aflatoxin M1. The quantification of aflatoxin M1is then performed by high performance liquid chromatography using fluorometric detection. This method was successfully carried out on naturally‐contaminated and spiked cheeses. Recoveries are about 75%. The limit of quantification is 0.020 μg of aflatoxin M1per kg of cheese. This method seems suitable for use in monitoring programmes for aflatoxin M1contamination in dairy products such as cheese.