Fe3O4 nanoparticles coated with chito-oligosaccharides (COS) were prepared in situ by a simple co-precipitation method through a mixing of iron ions (Fe3+ and Fe2+) and COS aqueous solutions followed by precipitation with ammonia. The impact of COS with different degree of polymerization (DP 10, 24 and 45) and degree of N-acetylation (DA) ∼ 24% and 50% (exhibiting high solubility) on the synthesis and physical properties of the coated magnetic nanoparticles was evaluated. Several advantages were found when the magnetic nanoparticles were prepared in the presence of the studied COS, such as: preparation of functionalized magnetic nanoparticles with narrower size distributions and, consequently, higher saturation magnetization (an increase of up to 22%); and an expressive increasing in the concentration of COS-coated magnetic nanoparticles (up to twice) in the cell viability test in comparison with pure Fe3O4 nanoparticles. Furthermore, among the analyzed samples, the magnetic nanoparticles coated by COS with DA ∼ 50% present a higher cytocompatibility. Our results allow envisioning various biomedical applications, valorizing the use of coated-magnetic nanoparticles for magnetic-field assisted drug delivery, enzyme or cell immobilization, or as a marker for specific cell tracking, among others.