Magnetite (FeO) nanoparticles (NPs) are attractive nanomaterials in the field of material science, chemistry, and physics because of their valuable properties, such as soft ferromagnetism, half-metallicity, and biocompatibility. Various structures of FeO NPs with different sizes, geometries, and nanoarchitectures have been synthesized, and the related properties have been studied with targets in multiple fields of applications, including biomedical devices, electronic devices, environmental solutions, and energy applications. Tailoring the sizes, geometries, magnetic properties, and functionalities is an important task that determines the performance of FeO NPs in many applications. Therefore, this review focuses on the crucial aspects of FeO NPs, including structures, synthesis, magnetic properties, and strategies for functionalization, which jointly determine the application performance of various FeO NP-based systems. We first summarize the recent advances in the synthesis of magnetite NPs with different sizes, morphologies, and magnetic properties. We also highlight the importance of synthetic factors in controlling the structures and properties of NPs, such as the uniformity of sizes, morphology, surfaces, and magnetic properties. Moreover, emerging applications using FeO NPs and their functionalized nanostructures are also highlighted with a focus on applications in biomedical technologies, biosensing, environmental remedies for water treatment, and energy storage and conversion devices.