The malolactic fermentation (MLF) led by Oenococcus oeni is an important technological step in high-quality wine production. The preparation of O. oeni starter with high viable count is one of the important prerequisites to ensure the smooth progress of the step. It was found that a certain amount of L-malic acid could effectively promote the growth of O. oeni, and the O. oeni cell density could be improved by optimizing the high-density culture conditions. The results showed that the optimal high-density culture conditions by orthogonal experiments were as follows: initial pH 5.1, inoculum 3% and L-malic acid addition 1 g/L. Under the optimal conditions, the cell density of O. oeni ES-1 was higher with (7.33±0.40)×109 CFU/ml. Combined with chemical neutralization method and semi-continuous culture method, the final cell density reached (1.67±0.11)×1010 CFU/ml, which was 11 times that of the control group. The optimization scheme of high-density culture obtained in this study could lay a foundation for the preparation of high quality local O. oeni starter.