This paper reports the preparation and characterization of thermosensitive poly(N-isopropylacrylamide) (PNIPAM)/magnetite nanoparticles in various conditions. The nanoprecipitation conditions address the impact of the temperature on PNIPAM/magnetite nanoparticle features due to the thermosensitive character of PNIPAM. Hybrid nanoparticles with desired features (size, size distribution, agglomeration, and release profile) are prepared by nanoprecipitation in non-solvent (acetone) at various temperatures. These nanoparticles are targeted as nanocarriers to deliver doxorubicin in breast cancer cells. Therefore, three temperatures, below the LCST (lower critical solution temperature), around the LCST, and above the LCST, were chosen as the main parameters within nanoprecipitation. Besides temperature, another major parameter drives the nanoparticles’ features: polymer solution concentration. In this regard, two variable parameters were used to study the characteristics of developed hybrid nanoparticles. After preparation, the hybrid nanoparticles were subjected to morphological and size distribution investigation by SEM and DLS. The doxorubicin loading and release measurements were also performed to reveal the behavior of the nanoparticles. Finally, the unloaded and loaded hybrid nanoparticles were biologically assessed within a cancer cells line (MCF7) in terms of biocompatibility, cancer cell viability, and cell morphology.