The invention relates to a biological de-acidification method of fruit wine, which comprises the following steps that: after being smashed, denucleated, juiced, 18 to 22 percent (w/w) sugar adjusted, adjusted with 100 to 120 mg/kg of sulfur dioxide and being added with yeast, the raw material fruit containing malic acid is undertaken the alcoholic fermentation with temperature being controlled; during the alcoholic fermentation process, the ratio of the beer is monitored, when the ratio is decreased to 1.015 to 0.990 (at the temperature of 20 DEG C), lactobacillus casei R35 which is previously activated and cultured is added; and when the total sugar content of the fermented beer is decreased to the setup value required for the wine, the fermentation is stopped. Through the biological de-acidification, the acidity of the fruit wine is reduced, the softness of the wine is improved, the flavor of the wine is improved, the quality of the fruit wine is improved, and the harm on the quality of the fruit wine caused by the chemical de-acidification can be prevented. The method has simple operation and low cost, is applicable to the large-scale population and application of different fruit wines, and has remarkable economic benefit.