Abstract: The thoroughly mixed sample of methyl ester of poly-ethoxyl fatty acid (abbreviated as MEE) was filtered by suction and centrifuged at high speed to separated into 2 layers, giving a clear supernatant upper layer and a turbid or solidified lower layer. The 2 layers were separated and collected for analytical study. Magnesium as a residual element from the catalyst was determined by FAAS. Polyethylene glycol (PEG) in both layers as a by-product in the synthesis of MEE was determined by the method of GB/T 5560-2003. Definite amount of lower layer was taken and its content of the by-product diester was determined by 1H NMR after hydrolysis and separation. It was found that residual amount of Mg2+ in the lower layer was 0.421 mg·g-1; amount of PEG found in the lower layer was 3.68%; and mole percentages of the by-product diester (PEG removed preliminarily) and MEE with high EO addition number were found to be 16% and 84% respectively. It was proposed that the difference in composition and content of the 2 layers was the cause of the layer separation phenomenon.