Abstract: In order to investigate carbonyl compound emission of methanol-diesel fuel and bio-diesel fuel, 14 sorts of different carbonyl compound from 3 different fuels (diesel, methanol-diesel and biodiesel) were investigated using High-performance Liquid Chromatography. The result showed the unregulated emissions from the two substitute fuels were greatly variable: 1) the overall specific emission of carbonyl compound increased. Compared with diesel, the carbonyl compound emission from Methanol-diesel increased 144.6%, and emission from biodiesel increased 67.5%; 2) formaldehyde was above 35.3% in the carbonyl compound emission under the testing condition; 3) In general, carbonyl compound increased as speed increasing or load decreasing when engine fueled methanol-diesel and biodiesel. The study provides reference for building carbonyl compound standards for using substitute fuels and matching the environment protection energy saving requirements.