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Biodiesels, which are produced through the transesterification reaction of fatty acids with alcohols, are promising clean alternative fuels in substitution of conventional diesel fuels. In this paper, a novel biodiesel, triethylene glycol monomethyl ether cottonseed oil monoester (TGMECOM), was developed. It was synthesized through transesterification of refined cottonseed oil and triethylene glycol monomethyl ether (TGME) with KOH as catalyst. Its chemical structure was characterized through FTIR, 1H NMR and GC-MS analyses. To investigate the engine performance of TGMECOM, tests on a two-cylinder DI diesel engine were conducted with different concentrations blended into diesel fuel. The results indicate that, compared to diesel fuel, TGMECOM has higher cetane number and oxygen content. For TGMECOM, the combustion timing of engine is advanced, ignition delay is shortened, and the combustion is improved.