Superparamagnetic nanoparticles have seen increased potential in medical and environmental applications. Their preparation is traditionally made by the coprecipitation method, with limited control over the particle size distribution. Microemulsion methods could be advantageous due to the efficient control of the size, shape, and composition of the nanoparticles obtained. Water-in-oil (W/O) microemulsions consist of aqueous microdomains dispersed in a continuous oil phase, stabilized by surfactant molecules. These work as nanoreactors where the synthesis of the desired nanoparticles takes place through a co-precipitation chemical reaction. In this work, superparamagnetic magnetite nanoparticles with average diameters between 5.4 and 7.2 nm and large monodispersity have been synthesized through precipitation in a W/O microemulsion, with Cetyl Trimethyl Ammonium Bromide (CTAB) as a main surfactant, 1-butanol as a cosurfactant, and with 1-hexanol as the continuous oily phase. The optimization of the corresponding washing protocol has also been established since a strict control is required when using these materials for bioapplications. Their applicability in those has been proved by their encapsulation in liposomes, being tested as signal enhancers for lateral flow immunoassays by using the affinity neutravidin-biotin model system. Due to their magnetic behaviour, they were also tested for magnetic separation. These novel materials have been found to be useful for analytical applications requiring high sensitivity and the removal of interferences.