In recent years, coinciding with the expansion of biofuel production, attempts have also been made to optimize production processes. In this study, Response Surface Methodology (RSM) was used to investigation the transesterification reaction of rapeseed oil for biodiesel production. Three main factors in order to convert triglycerides into fatty acid methyl esters (FAME) were applied according to a central composite design. These factors were catalyst concentration (NaOH), reaction temperature and time. The yield of methyl ester as the first response was determined using NMR method. The second response was the commercial cost of production. The results showed that the best conditions for producing biodiesel in constant the molar ratio of 1: 6 oil: methanol were the temperature of 47.27 oC, NaOH concentration of 1.24 %wt/wt and reaction time of 30 min. At these optimum conditions, the yield of methyl ester and cost of production is 77.67% and 67 ¢, respectively. Also, some chemical and physical properties of biodiesel were compared with petro-diesel fuel. According to the results, biodiesel fuel is a suitable substitute for petro-diesel fuel.