The two-step catalyzing process for biodiesel production from waste vegetable oil was assisted by both co-solvent and microwave irradiation. Central composite design (CCD) was employed to optimize the reaction conditions. Optimal reaction conditions of the first step were alcohol to oil molar ratio of 9:1, catalyst (H₂SO₄) amount 1 wt%, reaction temperature 333 K, and reaction time 7.5 minutes; while for the second step, optimal reaction conditions were alcohol to oil molar ratio 12:1, catalyst (NaOH) amount 1 wt%, reaction temperature 333 K, and reaction time 2.0 minutes. The total reaction time was 9.5 min and the conversion rate of fatty acid methyl esters (FAMEs) achieved was 97.4%. The total reaction time was shorter than previous studies. Therefore, the co-solvent and microwave assisted two-step catalyzing process has a potential application in producing biodiesel from waste vegetable oil.