Magnetic nanoparticles (MNPs) have unique physical and chemical properties, such as high surface area to volume ratio and size-related magnetism, which are completely different from their bulk materials. Benefiting from the facile synthesis and chemical modification strategies, MNPs have been widely studied for applications in nanomedicine. Herein, we firstly summarized the designs of MNPs from the perspectives of materials and physicochemical properties tailored for biomedical applications. Magnetic particle spectroscopy (MPS), first reported in 2006, has flourished as an independent platform for many biological and biomedical applications. It has been extensively reported as a versatile platform for a variety of bioassays along with the artificially designed MNPs, where the MNPs serve as magnetic nanoprobes to specifically probe target analytes from fluid samples. In this review, the mechanisms and theories of different MPS platforms realizing volumetric- and surface-based bioassays are discussed. Some representative works of MPS platforms for applications such as disease diagnosis, food safety and plant pathology monitoring, drug screening, thrombus maturity assessments are reviewed. At the end of this review, we commented on the rapid growth and booming of MPS-based bioassays in its first 15 years. We also prospected opportunities and challenges that portable MPS devices face in the rapidly growing demand for fast, inexpensive, and easy-to-use biometric techniques.