Specific ssDNA aptamers for the antibiotic florfenicol (FF) were developed from an enriched nucleotide library using magnetic beads-based SELEX (Systematic Evolution of Ligands by EXponential enrichment) technique with high-binding affinity. After 12 rounds of selection, thirty-six sequences were obtained that were then divided into five major families, according to the primary sequence similarity. Binding affinity analyses of three fluorescently tagged aptamers belonging to different families demonstrated that the dissociation constants (Kd) were in the low nanomolar range (Kd = 52.78–211.4 nmol L−1). Furthermore, to verify the potential application of the aptamers, a fluorescent aptasensor was fabricated for detecting the FF residue in raw milk samples based on the energy transfer between graphene oxide as the acceptor and fluorescently tagged FF-specific aptamer as the donor. Under optimal conditions, the aptasensor displayed a wide linear range from 5 to 1200 nmol L−1 and a detection limit of 5.75 nmol L−1 with excellent selectivity in milk. The recovery rate in the milk was between 101%±0.14% and 110%±2.8%, indicating high accuracy. This fluorescent aptasensor possessed considerable potential for rapid analysis of FF in raw milk because of its simplicity of detection. Moreover, the interaction between the aptamer and FF was studied using molecular modeling.

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