Fleroxacin (FLE) is a broad-spectrum fluoroquinolone antibiotic widely used in animal husbandry, veterinary medicine and aquaculture. Eating animal-derived foods with FLE residues can cause allergies, poisoning or drug resistance. The water-soluble QDs (CdSe/ZnS) and anti-FLE monoclonal antibody (mAb) were used to prepare a fluorescent probe by the method of N-(3-dimethylaminopropyl)-N′-ethylcarbodimide hydrochloride (EDC) activation. The fluorescent probe was characterized by dynamic light scattering (DLS). The better bioactivity and stability of the fluorescent probe was obtained under the pH value of 8.0, the molecule molar ratio of EDC (1 : 2000) and anti-FLE monoclonal antibodies (1 : 10). The control line (C line) and test line (T line) of a nitrocellulose (NC) filter membrane were sprayed with SPA (0.05 mg mL⁻¹) and FLE-OVA (1.4 mg mL⁻¹) solutions with optimal concentration, respectively. A novel method of fluorescent immunochromatographic assay based on quantum dots (QDs-ICA) in this work exhibited good accuracy, reproductivity and excellent specificity under the optimal experimental conditions. Compared with the traditional method for the visual detection of FLE, the developed QDs-ICA can successfully determine FLE residues in pork meat with a better cut-off value of 2.5 ng mL⁻¹. The QDs-ICA could be adapted for the rapid preliminary detection of FLE residues in pork meat for the first time.