A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop assays similar to the enzyme-linked immunosorbent (ELISA) assay to determine and quantify florfenicol (FF) in real food samples such as liquid milk and salmon muscle is presented here. The nanoMIPs were synthesized by a solid-phase approach with an immobilized FF (template) and characterized using dynamic light scattering, a SPR-2 biosensor system and transmission electron microscopy. Immobilization of nanoMIPs was conducted by preparing a homogenous solution of FF-nanoMIPs in water mixed with polyvinyl alcohol (PVA) 0.2% (w/v) in each well of a microplate. The detection of florfenicol was achieved in competitive binding experiments with a horseradish peroxidase−florfenicol (FF-HRP) conjugate. The assay made it possible to measure FF in buffer and in real samples (liquid milk and salmon muscle) within the range of 60−80 and 90-100 ng/mL, respectively. The immobilized nanoMIPs were stored for six weeks at room temperature and at 5 • C. The results indicate good signal recovery for all FF concentrations in spiked milk samples, without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELISA.with florfenicol (FF) being the main antibiotic used (52%), followed by oxytetracycline, flumequine and oxolinic acid (44%, 1% and 1%, respectively) [5,6]. FF, a fluorinated derivative of thiamphenicol, is a new generation of fenicol drugs [7]. Apart from in the aquaculture industry, FF is widely used as chloramphenicol alternative to prevent and treat bacterial diseases in pigs [8], bovine [9], poultry [10] and animal edible tissues [11]. Several countries adopted maximum permissible residue limits (MRLs) of antibiotics in foods of animal origin, such as meat, milk and eggs [12]. In Chile, the maximum permissible residue limit of FF in muscle and skin of salmon is 1000 µg/kg [13]. At present, no MRL has been set for FF in milk. It is however of vital importance to develop a reliable method for FF analysis in milk products to avoid potential harmful effects to consumers [14]. Therefore, there is a demand for relevant sensitive, selective, fast and inexpensive analytical protocols from legislation, health authorities and companies operating in the food market [15]. Various analytical techniques have been reported for the analysis of FF, including high performance liquid chromatography, gas chromatography, liquid chromatography-Tandem mass spectrometry [16], gas chromatography-Mass spectrometry [17], and enzyme-linked immunosorbent assay (ELISA) [18]. The manufacture of molecular imprinting polymers (MIP) can be very valuable for those selective and sensitive methods that are needed. Molecular impression is a technique for producing in a polymer matrix selective binding sites that recognize a particular molecule. These polymers are very sta...