This study investigated the characterization of cobalt ferrite nanoparticles (CoFe2O4) prepared by the pulsed laser deposition (PLD) method, using various techniques such as energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and in vitro drug release test (IVRT). It also evaluated the toxicity of these nanoparticles on liver, spleen, heart, and kidney tissues using a mouse model. CoFe2O4 nanoparticles were synthesized from an aqueous solution containing 1mg of CoFe2O4 powder dissolved in DMSO and RPMI serum-free media. XRD analysis revealed the characteristic peaks of the spinel lattice of cubic-shaped cobalt ferrite nanocrystals, with an average crystallite size of 12 nm calculated using Scherrer’s formula. SEM and EDX were used to study the surface morphology and chemical compositions, respectively. The in vivo experiments determined the LD50 of CoFe2O4 to be 3000 mg/kg body weight. Administration of CoFe2O4 nanoparticles at a dosage of 75 mg/kg body weight for 14 days did not cause any changes to the histological structure of the tissues in the mouse model, indicating their potential biocompatibility.