Reaction temperature, alcohol/oil molar ratio, type and amount of catalyst, reaction duration, and the water content in free fatty acid are the most important parameters in biodiesel production by the transesterification method. Thus, it is important to define the optimum values of these parameters to achieve the EN 14214 standards. In this study, optimum levels of the variables affecting the yield of tobacco seed oil methyl ester (TSOME) for KOH and NaOH catalysts are investigated. Reaction temperatures of 40, 50, and 60 degrees C, alcohol/oil molar ratios of 1:4, 1:5, 1:6, and 1: 10, and the addition of 0.5, 1, and 1.5% KOH catalyst of tobacco seed oil (TSO) were chosen as parameters. Effects of these parameters on kinematic viscosity, flash point, and freezing points are also investigated. The effect of the reaction duration on the yield of ester is also studied.