Hepatitis A virus (HAV) continues to be the leading cause of viral hepatitis. HAV outbreaks have been linked to the consumption of milk, but methods for HAV detection in milk are very limited. We developed a method to concentrate HAV in milk using protamine-coated iron oxide (Fe3O4) magnetic nanoparticles (PMNPs). In this study, protamine was covalently coated on the surface of the MNPs (20-30 nm) by a three-step chemical reaction. The successful linkage of protamine to the MNPs was confirmed by Fourier transform infrared spectroscopy (FTIR), zeta potential, and transmission electron microscopy (TEM). When used for concentrating HAV from 40 mL of milk, 50 μL of PMNPs were added to the sample and mixed for 20 min by gentle rotation, followed by a magnet capture for 30 min. The captured PMNPs were washed with glycine buffer (0.05 M glycine, 0.14 M NaCl, 0.2% (v/v) Tween 20, pH 9.0) and HAV RNA was extracted using the QIAamp MinElute Virus Spin Kit and quantified by real-time RT-PCR. The method showed a detection limit of 8.3 × 100 PFU of HAV in milk. The whole concentration procedure could be completed in approximately 50 min. The developed method was simple, inexpensive, and easy-to-perform.