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However, their biological instability and reliance on labor\u2010intensive chemical conjugation hinder practical high\u2010throughput screening of food contaminants. To address this, we report a rapid, label\u2010free luciferase\u2010based platform for quantifying lomefloxacin (LOM) in food. The assay uses an engineered immunoprobe: a high\u2010affinity single\u2010chain variable fragment antibody (3G9\u2010scFv) specific to LOM, combined with Nanoluciferase (NLuc) as an intrinsic signal generator without covalent labeling. NLuc catalyzes furimazine oxidation, producing stable bioluminescence inversely proportional to LOM concentration. This label\u2010free design eliminates conjugation variability and simplifies protocol execution. Smartphone\u2010based luminescence measurement enables sensitive field detection, with a limit of quantification of 0.12\u00a0ng\/mL. Furthermore, homology modeling and molecular docking elucidated the structural basis for the dual functionality of 3G9\u2010scFv, validating the rational use of unlabeled enzymatic tracers. The visual detection limit was determined to be 1\u00a0ng\/mL. Recovery rates ranged from 79.0% to 113.0%, with coefficients of variation (CVs) between 2.3% and 10.9%, indicating high accuracy and reproducibility. This system offers a novel rapid detection platform for monitoring hazardous residues in food safety.<\/jats:p>","DOI":"10.1002\/smll.75027","type":"journal-article","created":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T14:52:21Z","timestamp":1786719141000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Label\u2010Free Bioluminescent scFv\u2010NLuc Fusion Probe Enabling a Smartphone\u2010Based Sensitive Immunoassay Platform Using Lomefloxacin as a Model Analyte"],"prefix":"10.1002","volume":"22","author":[{"given":"Mingyue","family":"Ding","sequence":"first","affiliation":[{"name":"National Reference Laboratory of Veterinary Drug Residues (HZAU) and MOA Key Laboratory For the Detection of Veterinary Drug Residues Huazhong Agricultural University  Wuhan China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Wen","sequence":"additional","affiliation":[{"name":"National 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