A novel biosensor for the highly sensitive detection of aflatoxin M(1) (AFM(1)) in milk was developed. This biosensor is based on surface plasmon-enhanced fluorescence spectroscopy (SPFS) which was advanced through the excitation of long range surface plasmons (LRSPs). In SPFS, the binding of fluorophore-labeled molecules to the sensor surface is probed with surface plasmons (SPs) and the emitted fluorescence light is detected. This approach takes advantages of the enhanced intensity of electromagnetic field occurring upon the resonant excitation of SPs which directly increases the fluorescence signal. For the detection of AFM(1), LRSP-enhanced fluorescence spectroscopy was combined with an inhibition immunoassay in which a derivative of AFM(1) was immobilized on the sensor surface and antibodies against AFM(1) were used as recognition elements. The developed biosensor allowed for the detection of AFM(1) in milk within 53min at concentrations as low as 0.6pgmL(-1). The achieved limit of detection was about two orders of magnitude lower than the maximum AFM(1) residue level in milk stipulated by the European Commission legislation.