The aim of this study was to evaluate the effects of fermentation conditions on the cumulative esterase activity of Oenococcus oeni (O.oeni) autochthonous strains in the Hexi Corridor region, particularly in the aromaticesters of Chardonnay dry white wine during the Malolactic Fermentation (MLF). Two O. oeni autochthonous strains, such as GF-2 and ZX-1, were preserved by the Gansu Key Lab Viticulture and Enology, and one commercial strain VP41 was used to test strains. The esterase activities of various carbon chain length substrates (C2, C4, C6) were detected in the simulated wine during MLF process. The fermentation conditions were selected to analyze the esterase characteristics produced by the O. oeni strains, including the initial pH value, ethanol concentration, SO2 addition, and fermentation temperature. A microvinification experiment was performed to explore the modification effect of the tested strains on the aroma quality of Chardonnay dry white wine. The esterase activities of O. oeni autochthonous strains were significantly higher than those of commercial strain VP41 under different pH values, where the maximum esterase activity of ZX-1 was about 63.42% higher than that of VP41. When the concentration of ethanol was 8%, all the tested strains produced the maximum esterase activity, indicating the O.oeni autochthonous strain had strong esterase producing ability. Under the different SO2 additions, the cumulative esterase activities of two O. oeni autochthonous strains were significantly higher than that of VP41 (P<0.05), while, the esterase activity of GF-2 was significantly higher than those of strain VP41 at 18 and 22 ℃ (P<0.05). Results of compound fermentation showed that the total esterase activity originated from ZX-1 was the highest, followed by GF-2 and VP41. There were different effects of the major and secondary factors on the esterase activity of each strain. An optimum condition was obtained for the esterase production of all tested strains: the ethanol concentration of 12%, pH value of 3.6, SO2 addition of 30 mg/L, and the fermentation temperature of 22 ℃. The highest esterase activity of ZX-1 was 620.97 mU/mL, indicating that a strong adaptability to wine habitat. In the microvinification of chardonnay dry white wine, esters were identified in the wine samples after MLF. There were richer variety aroma and better fragrance persistence in the wines that fermented by autochthonous strains GF-2 and ZX-1, compared with the commercial strain VP41. Both O.oeni autochthonous and commercial strains can successfully complete MLF, especially ZX-1 has strong esterase production capability, depending mainly on the fermentation conditions. Therefore, the ZX-1 can be used to effectively improve the content of fruit and floral aroma compounds in the Chardonnay dry white wine, thereby to enhance the regional microbial terroir characteristics of wines. The O. oeni autochthonous strain ZX-1 was more suitable to be used as the MLF starter of dry white wine in the Hexi Corridor of Gansu Province.