This research work focuses on the production of biodiesel from refined cottonseed oil via alkaline-transesterification process using sodium hydroxide catalyst and methanol. Optimization was carried out using a 2 3factorial design experiment, three variables namely mole ratio of methanol to oil, catalyst concentration and reaction temperature were studied at both high and low levels. The results obtained during the characterization of refined cottonseed oil include; Saponification Value (189.0 mgKOH/g), Acid Value (1.02 mgKOH/g), Free Fatty Acid (0.51%), Iodine Value (111.2 g/100g), Specific Gravity (0.920), Refractive Index (1.468). These results show the suitability of the oil for the production of biodiesel. Production yield and purity of biodiesel were used to verify the optimization. Results obtained showed that mole ratio of methanol to oil and catalyst concentrations were the most important variables affecting the yield of biodiesel. Optimum methyl ester yield of 96.23% and a concentration of 99.54% were gotten at a temperature of 40 °C, mole ratio of methanol to oil of (6:1) and catalyst concentration of 0.5 wt% at a constant reaction time of 90 minutes.