This study investigated the transesterification of waste cooking oil (WCO) with methanol in the presence of potassium hydroxide as the catalyst. A semi-industrial pilot of microreactor with 50 tubes with a diameter of 0.8 mm was used to produce 5 L/h biodiesel. Initially, the acidity of waste cooking oil was reduced to less than 1 mg KOH/g oil by using methanol at 60C in the presence of 1% sulfuric acid. Using Box-Behnken design method, the effects of methanol to oil molar ratio (6:1–12:1), catalyst weight (0.5–1.5 wt %), and reaction temperature (55–65C) were studied. The methanol to oil molar ratio of 9.4:1, the catalyst concentration of 1.16 wt %, and the reaction temperature of 62.4C was achieved under optimum condition. Finally, the effect of reaction time (30–120 s) was examined at the optimum condition. The highest level of biodiesel purity or fatty acid methyl esters % (FAMEs %) was 98.26%. In addition, the properties of produced biodiesel were determined and compared with those of the standard ASTM D6751.