Abstract A new method for highly sensitive detection of methimazole was developed based on the fluorescence resonance energy transfer (FRET) between acridine orange (AO) and silver nanoparticles (AgNPs), in which AO acts as a donor and AgNPs as an acceptor. The fluorescence of AO molecules was severely quenched when they were attached to the surface of AgNPs by electrostatic attraction. Upon the addition of methimazole, AO molecules were detached from NPs because of strong adsorption of methimazole on the surface of AgNPs. Under optimum conditions, the enhanced fluorescence intensity displayed a linear relationship with the concentration of methimazole in the range of 8.0×10−9–3.75×10−7M, with a limit of detection of 5.5nM. The method was applied for the determination of therapeutic levels of methimazole in human plasma samples with satisfactory results.