Biosensors are analytical devices that use biological interactions to detect and quantify single molecules, clinical biomarkers, contaminants, allergens, and microorganisms. By coupling bioreceptors with transducers, such as nucleic acids or proteins, biosensors convert biological interactions into electrical signals. Electrochemical and optical transductions are the most widely used methods due to their high detection capability and compatibility with miniaturization. Biosensors are valuable in analytical chemistry, especially for health diagnostics, as they offer simplicity and sensitivity. Despite their usefulness, challenges persist in immobilizing biorecognition elements on the transducer surface, leading to issues such as loss of sensitivity and selectivity. To address these problems, the introduction of nanomaterials, in particular magnetic nanoparticles (MNPs) and magnetic beads, has been implemented. MNPs combine their magnetic properties with other interesting characteristics, such as their small size, high surface-to-volume ratio, easy handling, and excellent biocompatibility, resulting in improved specificity and sensitivity and reduced matrix effects. They can be tailored to specific applications and have been extensively used in various fields, including biosensing and clinical diagnosis. In addition, MNPs simplify sample preparation by isolating the target analytes via magnetic separation, thus reducing the analysis time and interference phenomena and improving the analytical performance of detection. The synthesis and modification of MNPs play a crucial role in adjusting their properties for different applications. This review presents an overview of the synthesis and surface modifications of magnetic nanoparticles and their contributions to the development of biosensors and bioassays for their applications across different areas. The future challenges of MNP synthesis and integration in assays are focused on their stability, multiplex detection, simplification and portability of test platforms, and in vivo applications, among other areas of development.