The invention provides a preparation method of a low-acidity fruit juice fermentation broth. The preparation method comprises the step of adding yeast sedimentation liquid into a fruit juice fermentation broth to be subjected to deacidification and clarification for primary deacidification to prepare a primary deacidification fermentation broth and the step of adding the yeast sedimentation liquid into the primary deacidification fermentation broth for deacidification again. Compared with the prior art, the preparation method of the low-acidity fruit juice fermentation broth has the following advantages that change of flavor substances cannot be brought, and the risk of storage stability cannot be increased; when inside and outside osmotic pressure is balanced, adsorption competition of a yeast cell on organic acids such as citric acid is large, and the adsorbing effect on kiwi fruit and citrus fermentation broths is obvious; the yeast cell can be recycled through washing, releasing and regeneration after adsorbing the organic acids, the brewer's yeast has the advantages of being rich in resource, easy to proliferate, easy to use and the like, and the preparation method can be applied in production practice.