Nanotechnology provides exciting new possibilities for advanced development of new analytical tools and instruments for bioanalytical applications. Magnetic nanoparticles (MNPs) have attracted much research interest in the past decade because they have good biocompatibility and can be readily separated from reaction mixtures with the aid of an external magnetic field. Heterogenic fluorescent immunoassays for determination of different analytes (antibiotics, pesticides, progesterone, aflatoxins, enterotoxins) using MNPs were developed. MNPs were prepared by thermal co-precipitation of Fe2+ and Fe3+ in aqueous solution. MNPs were functionalized with (3-aminopropyl) triethoxysilane. Two different analyzes were made for proving the successful modification of MNPs – FTIR and TEM. The corresponding antibodies were immobilized on modified MNPs. Then corresponding antigen - fluorescent dye conjugates were synthesized. The next step was purification of the resulting conjugates by gel filtration chromatography and proving them by UV-Vis and Fluorescence analyses. The 9 different separate competitive MNPs based immunoassays were developed – for penicillin, sulfonamide, tetracycline, progesterone, aflatoxin M1 and B1, enterotoxin A, paraoxon and dichlorvos. Then multiplex fluorescent immunoassay device based on magnetic nanoparticles for determination of penicillin, sulfonamide and tetracycline in milk sample was developed. The device ensured fast, selective and cheap antibiotic analysis. Very promising analytical characteristics for all three antibiotics were received - very low LODs and wide linear range. Practical applications: The multiplex fluorescent immunoassay device based on magnetic nanoparticles for determination of penicillin, sulfonamide and tetracycline in milk sample was developed. Device ensured fast, selective and cheap antibiotic analysis. Very promising analytical characteristics for all three antibiotics were received - very low LODs and wide linear range.