Quantitative and sensitive measurements of clinically relevant pathogens and biomarkers from biological samples would be invaluable for early diagnosis of diseases. In modern era, magnetic nanoparticles (MNPs) are found to be of great interest due to their unique features. Specifically, biological samples exhibit negligible magnetic responses, which enables MNPs for highly sensitive measurements on minimally processed samples. The aim of this chapter is to provide an insight to the applications of MNPs for the in vitro detection of target ligands based on two detection platforms: volumetric- and surface-based biosensing platforms. Different immunoassay methods such as search coil-based biosensors, giant magnetoresistance (GMR) biosensors and Hall sensors are reviewed. We also discuss immunoassay strategies associated with MNPs such as the sandwich and competitive bioassays. Applications of MNPs for the detection of foodborne pathogens, viruses and heavy metals are also highlighted.