Thymol is the major antimicrobial and bioactive constituent found in thyme (Thymus vulgaris) essential oil. In this study, it was aimed to determine the parameters for fabrication of thymol loaded chitosan nanoparticles with optimum encapsulation efficiency, zeta potential, and particle size properties using a two-stage emulsion-ionic gelation approach. For this purpose, temperature (25-45 °C) and chitosan (3-6 mg/mL), thymol (3-6 mg/mL), Tween 80 (3-6 mg/mL) and TPP (0.15-0.75 mg/mL) concentrations were studied as optimization parameters by applying the numerical point prediction method. The results showed that, it was determined that minimum particle size, maximum zeta potential and maximum encapsulation efficiency. The results showed that the particle size, zeta potential and encapsulation efficiency of the chitosan nanoparticles could be controlled by processing conditions. Additionally, this study was focused on optimization of these parameters with factorial design (FD) in nanoencapsulation of Thymol. The optimized formulation of nanoparticles was selected on the basis of the criteria of attaining the minimum particle size, maximum zeta potential, and maximum encapsulation efficiency could be optimized when 42 °C temperature, chitosan rate 3 mg/mL, Thymol rate 5.9 mg/mL, Tween 80 rate 3 mg/mL and TPP rate 0.75 mg/mL were applied.