A one-step generic chemiluminescence competitive direct enzyme immunoassay (CL-cdELISA) based on a single-chain variable fragment (scFv)–alkaline phosphatase (AP) fusion protein (CL-cdELISAscFv–AP) was developed. It was capable of detecting 20 targeted fluoroquinolones (FQs) in fish and shrimp matrices within 40 min below the maximum residue levels (MRLs). In the optimized generic assay, the scFv–AP fusion protein in combination with a norfloxacin–ovalbumin conjugate (NOR–OVA) showed 50% binding inhibition (IC50) at 0.15 ± 0.01 μg kg?1 for NOR in 0.01 M phosphate-buffered saline (PBS), indicating that it is seven times as sensitive as the corresponding competitive direct enzyme immunoassay (cdELISAscFv–AP), and the linear response range of the assay was extended from 0.04 to 1.08 μg kg?1. The limits of detection (LODs) of the assay for NOR were 0.017 μg kg?1 in shrimp and 0.018 μg kg?1 in fish, and the LODs inferred from the cross reactivity (CR) ranged from 0.013 μg kg?1 for ciprofloxacin (CIP) to 4.19 μg kg?1 for trovafloxacin (TRO); the recoveries of the three representative antibiotics norfloxacin, flumequine (FLU) and sarafloxacin (SAR) from spiked fish and shrimp samples varied from 72.50 to 118.50% and the mean coefficients of variation for the inter-assay and intra-assay were 6.4% and 9.2%, respectively. Further validation of CL-cdELISAscFv–AP with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed that the assay was a reliable screening tool for the detection of FQs in fish and shrimp.