Novozym-435-catalysed esterification of caprylic acid, capric acid and oleic acid with glycerol for the synthesis of medium- and long-chain triglycerides (MLCT) in vacuum and solvent-free system was investigated in this study. Response surface methodology with a three-level, four-factorial design was applied to optimise the enzymatic esterification for the synthesis of MLCT. The optimum conditions were as follows: reaction temperature 90 °C, 4.80 wt% enzyme load (relative to the weight of total substrates) and substrate molar ratio (fatty acids/glycerol) of 3:1 and 12.37 h. Under above-mentioned conditions, Triglycerides (TG) yield, MLCT and the residual free fatty acids (FFA) content in the product were 93.54%, 72.19% and 4.21%, respectively. The content of caprylic acid, capric acid and long-chain fatty acids of TG was 24%, 10% and 66%, respectively. Novozym 435 in the study showed no selectivity for the different fatty acids and also could be used 14 times without obvious loss of enzyme activity.