Biodiesel was produced through transesterification of refined cotton seed oil with methanol and potassium hydroxide (KOH) as a catalyst using batch mode. The physicochemical properties of cotton seed oil and biodiesel as an alternative fuel for diesel engine was characterized through ASTM standards for fuel tests. The functional groups of the biodiesel were investigated using Fourier transform infrared spectroscopy. Influence of key parameters like reaction temperature, reaction time, catalyst concentration and methanol/oil molar ratio were determined using batch mode. These process parameters were optimized using response surface methodology (RSM) and analysis of variance (ANOVA). The significance of the different process parameters and their combined effects on the transesterification efficiency were established through a full factorial central composite design. The results obtained are in good agreement with published data for other vegetable oil biodiesel as well as various international standards for biodiesel fuel. An optimum yield of 96% was achieved with optimal conditions of methanol/oil molar ratio, 6:1; temperature, 55°C; time, 60min; and catalyst concentration, 0.6%. This investigation has shown that cotton seed oil from Nigeria can be used to produce biodiesel.