Nanoparticles have gained increased attention due to the prospection of drug delivery at target sites, thus limiting the systemic effects of the drugs. Their efficiency was further improved by adding special carriers such as magnetite (Fe3O4). It is one of the extensively used oxides of iron for both pharmaceutical and biomedical applications owing to its ease of preparation and biocompatibility. In this work, Gemcitabine magnetic nanoparticles were prepared using Fe3O4 and chitosan as the primary ingredients. Optimization was accomplished by Box–Behnken Design and factor interactions were evaluated. The desirability function approach was made to enhance the formulation concerning particle size, polydispersity index, and zeta potential. Based on this, optimized magnetic nanoparticles (O-MNP) were formulated with 300 mg of Fe3O4, 297.7 mg of chitosan, and a sonication time of 2.4 h, which can achieve the prerequisites of the target formulation. All other in vitro parameters were found to be following the requirement. In vitro cytotoxic studies for O-MNP were performed using cell cultures of breast cancer (MCF-7), leukemia (THP-1), prostate cancer (PC-3), and lung cancer (A549). O-MNP showed maximum inhibition growth with MCF-7 cell lines rather than other cell lines. The data observed here demonstrates the potential of magnetic nanoparticles of gemcitabine in treating breast cancers.