Ochratoxin A (OTA) is a mycotoxin produced by filamentous fungi of the genus Aspergillus and Penicillium that presents carcinogenic, teratogenic and nephrotoxic properties. In this work, we have developed, characterized and applied an immunoassay methodology comprised of magnetic nanoparticles (MNPs) as platform for immobilizing bioactive materials incorporated into a microfluidic system for rapid and sensitive quantification of Ochratoxin A (OTA) in apples (Red Delicious) contaminated with Aspergillus ochraceus. The sensor has the potential for automation and the detection of OTA was carried out using a competitive indirect immunoassay method based on the use of anti-OTA monoclonal antibodies immobilized on 3-aminopropyl-modified MNPs. The total assay time into the microfluidic competitive immunosensor was 16 min, and the calculated detection limit was 0.05 µg kg(-1). Moreover, the intra- and inter-assay coefficients of variation were below 6.5%. The proposed method can be a very promising analytical tool for the determination of OTA in apparently healthy fruits post-harvest and for its application in the agricultural industry.