The aim of the present work was to develop a sensitive and selective method for identification and quantification of fumonisin B1 (FB1) in milk. FB1 was isolated from milk, by a single step immunoaffinity column and was detected using liquid chromatography coupled with tandem mass spectrometry in positive electrospray ionization (ESI+). The analysis were carried out in multiple reaction monitoring (MRM) mode using the two main product ions. The good performances of the proposed method can assure a correct fumonisin detection in milk even at relatively low concentrations.