A fluorescence polarisation immunoassay (FPIA) using a specific polyclonal antiserum for the detection of maduramicin (MD) was developed and optimised. The polyclonal antiserum was produced against MD linked to bovine serum albumin. Fluorescein-labelled MD (tracer) was synthesised by N-hydroxysuccinimide active ester method and purified using thin layer chromatography. The developed FPIA for MD had a dynamic range from 0.01 to 5.6 μg mL−1 with an IC50 value of 0.16 μg mL−1 and a limit of detection of 0.002 μg mL−1. Recoveries from chicken muscle, fat and egg samples spiked at 0.25, 5 and 10 μg g−1 levels were 82–130%. The FPIA results from analysis of incurred residues in chicken muscle samples showed that the simple procedure is viable.