In this study, the influence of moisture and acids on the content of fatty acid methyl esters and triacetin in the interesterification reaction by using 1M potassium tert-butoxide in tetrahydrofuran as a homogeneous catalyst was investigated. During the interesterification reaction, the presence of moisture causes a larger negative effect than free fatty acids or acetic acid. The highest content of fatty acid methyl esters (77.6 wt%) and triacetin (17.4 wt%) was achieved at methyl acetate to oil molar ratio (MAOMR) 60 and catalyst to oil molar ratio (COMR) 0.05 by using raw materials with moisture and acids content no larger than 0.005% and 0.08%, respectively. During the interesterification reaction, the excess of the catalyst caused the formation of two-layer and its proceeds only by using high-quality raw materials. This fact has never been mentioned in the literature. The upper layer is biodiesel, the lower layer consists of glycerol and glycerol acetates (mono-, di-, and triacetin). The COMR has a more significant effect on the formation of the biodiesel and glycerol acetates layers than MAOMR. The highest yield of glycerol acetates layer (19.4%) has been formed by using MAOMR 60 and COMR 0.3.