Lactoferrin is a globular protein-based biopolymer which has many applications in pharmacy and food industry. In this study, human lactoferrin nanoparticles were synthesized by thermal treatment technique. Taguchi array design was employed to minimize the size of nanoparticles. To gain as this purpose, four main factors with four diferent levels involved as: lactoferrin concentration (1–4 mg/mL), pH values (6–9), heating rate of (2–5 °C/min) and fnal temperature of (60–90 °C) were investigated. In the following, prepared nanoparticles were characterized by dynamic light scattering, Fourier transform infrared spectroscopy, scanning electron microscopy, and atomic force microscopy analysis. Analysis of variance showed that lactoferrin concentration with the contribution percentage of 45% is most important in the preparation of nanoparticles. Also, optimal conditions for synthesized nanoparticles by thermal treatment technique were: LF concentration of 3 mg/mL, pH 8, heating rate of 3 °C/min and fnal temperature of 90 °C to achieve particle size of 188.8 nm, polydispersity index of 0.317 and zeta potential of −52.2 mV.