A procedure for separation and preconcentration of tetracyclines from human serum samples involving magnetic dispersive micro-solid phase extraction was proposed. The extraction efficiency of different tetracyclines was improved with the use of the surfactant coated Fe3O4 magnetic nanoparticles. Sorption mechanism was presented, and the potential use of magnetic Fe3O4 nanoparticles coated with different surfactants for tetracyclines adsorption was demonstrated for the first time. The procedure involved nanoparticle floating in a liquid sample phase for analyte extraction followed by elution and determination by high performance liquid chromatography with diode array detection. Influence of the main involved parameters was studied, the system was dimensioned accordingly. The analytical curves were linear in the ranges of 0.25–10 mg L−1 for tetracycline and 0.10–10 mg L−1 for oxytetracycline or doxycycline. Limits of detection were estimated (IUPAC, 3 concept) as 0.08 mg L−1 for tetracycline, and 0.03 mg L−1 for oxytetracycline or doxycycline. The proposed procedure proved to be fast (10 min), simple (two stages), inexpensive (10 mg of nanoparticles) and was applied to human serum samples. Unlike previously synthesized nanoparticles for tetracyclines separation, the surfactant-coated Fe3O4 nanoparticles can be easily prepared with widely available and low-cost reagents. Moreover, elution of the analytes was accomplished in absence of organic solvents by an aqueous chelating agent solution.