Magnetic nanoparticles (MNPs) have gained great attention for their remarkable physical and chemical properties in developing biosensors for different applications, such as environmental, clinical and biological. MNPs can be incorporated in the transducers or be dispersed into specimens, which are attracted by an external magnetic field to the active detection surface of biosensors. In this chapter, the synthesis, properties and characterization of MNPs for biosensor applications are discussed, together with recent applications of functionalized MNPs in such biosensor applications. Electrochemical, optical, piezoelectric and magnetic field based biosensors are discussed, considering their analytical properties, as well as enzyme-based biosensors, such as glucose oxidase and cholesterol oxidase-based biosensors.