The production of esters is largely influenced by functional gene expressions and the nutrition status of grape must. In this study, the effects of unsaturated fatty acids (UFAs) mixture (including linoleic acid, oleic acid and linolenic acid) on volatile esters of wine fermented with T73::ATF1 (overexpressing ATF1-encoded alcohol acetyltransferases) and T73::EEB (overexpressing EEB1-encoded acyl-CoA: ethanol O-acyltransferases) were investigated in alcoholic fermentation, respectively. The inoculation of T73::ATF1 into UFAs added juice significantly increased acetate esters production (ethyl acetate, isoamyl acetate and phenylethyl acetate) compared to in the control juice. The promotion on ethyl esters biosynthesis was also found in T73::EEB1 when supplemented with low level of UFAs, but the function became inhibition with the further increment of UFAs concentration. The levels of higher alcohols, medium fatty acids and the major genes expression (ADH1, BAT1, PDC1, ACC1, FAS1 and IAH1) were changed and varied among the both strains. These results indicated that UFAs can modulate the effect of overexpressing ATF1 and EEB1 on esters biosynthesis by influencing the substrates availability. The combination of increasing ATF1 and EEB1 expressions and modulating UFAs content is a potential means to increase the esters production and improve the aromatic quality of wines.