Superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as cutting-edge materials, garnering increasing attention in recent years within the fields of chemical and biomedical engineering. This increasing interest is primarily attributed to the distinctive chemical and physical properties of SPIONs. Progress in nanotechnology and particle synthesis methodologies has facilitated the fabrication of SPIONs with precise control over parameters such as composition, size, shape, stability, and magnetic response. Notably, these functionalized materials exhibit a remarkable surface-area-to-volume ratio, biocompatibility, and, most importantly, they can be effectively manipulated using external magnetic fields. Due to these exceptional properties, SPIONs have found widespread utility in the medical field for targeted drug delivery and cell separation, as well as in the chemical engineering field, particularly in wastewater treatment. Magnetic separation techniques driven by magnetophoresis have proven to be highly efficient, encompassing both high-gradient magnetic separation (HGMS) and low-gradient magnetic separation (LGMS). This review aims to provide an in-depth exploration of magnetic field gradient separation techniques, alongside a comprehensive discussion of the applications of SPIONs in the context of drug delivery, cell separation, and environmental remediation.