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A simple, sensitive and specific chemiluminescence enzyme immunoassay based on biotin–streptavidin system (BASA-CLEIA) for diclazuril was established, which was about 2 and 4 times more sensitive than the traditional chemiluminescence enzyme immunoassay (CLEIA) and enzyme-linked immunosorbent assay. In the optimized generic assay, the half maximal inhibitory concentration (IC50) of the BASA-CLEIA for diclazuril was 0.48 μg/kg. The limit of detection for diclazuril was 0.02 μg/kg. This method showed no cross reactivity (<0.01%) with toltrazuril, robenidine hydrochloride, nicarbazin, halofuginone, amprolium, monensin, and maduramycin. In chicken muscles, the average recoveries ranged from 90.3% to 120.4%. A further comparison between the developed BSAS-CLEIA and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for real samples showed good correlation (p < 0.01). The results indicated that the simple, sensitive and specific BASA-CLEIA method could be used for the analysis of diclazuril in real samples.