摘要:In this study, we carried out an amplified luminescent proximity homogeneous assay (AlphaLISA) to detect sulfonamides (SAs) antibiotic residues in plasma, milk, pork, chicken, and fish. The SAs AlphaLISA method can detect 13 SAs with half‐inhibitory concentration (IC50) 2.11–29.77 ng/ml. The detection level of those SAs was 0.3–41.12 ng/ml in matrices, which satisfied the maximum residue limit (MRL) of the European Union, United States, and China. Our recoveries are in the range of 88% to 116.8% with a coefficient of variation less than 9.3% for different spiked food samples. We observed a good correlation between the AlphaLISA and liquid chromatography–tandem mass spectrometry (LC‐MS/MS) with blood samples from injected rabbits. The established AlphaLISA method provided a no‐washing, rapid, high‐throughput screening tool for SAs in food quality control, which is suitable for small‐volume samples.