Immunoglobulin Y (IgY) is derived from egg yolk and has been identified as a cheap and high-yield immunoreagent. The application of IgY in immunoassays for the detection of chemical contaminants in food samples has rarely been reported. In this work, we describe a rapid and sensitive fluorescence polarization immunoassay (FPIA) for valnemulin (VAL) using IgY which was produced using a previously prepared immunogen. Three fluorophore-labeled VAL tracers were synthesized and the sensitivity of the best tracer (VAL-DTAF) in the optimized FPIA with antibody IgY100 demonstrated an IC50 value of 12 ng mL(-1) in buffer. After evaluation of several extraction procedures, acidified acetonitrile was selected to extract VAL from swine tissue. The recoveries of VAL in spiked swine tissue at three levels (50, 100, and 200 μg kg(-1)) were higher than 79% with coefficients of variation (CVs) lower than 12%. The limit of detection (LOD) of the FPIA in swine tissue was 26 μg kg(-1) and was lower than the maximum residue limit (MRL) of VAL set by the European Union. The study showed that IgY could be a good substitute for IgG when developing a high-throughput assay for chemical residues.