The invention discloses a preparation method of low-cholesterol bifidobacterium yoghurt. The preparation method includes that composite leavening agent is prepared firstly, fresh milk is sterilized in preliminary mode and degreased through high-speed centrifugal separation, cholesterol milk is removed, after sterilization, mixed culture is performed according to a raw material formula of 88-92% of fresh milk, 4-6% of water, 1.8-2.2% of white granulated sugar and 2.8-3.2% of composite leavening agent, preliminary fermentation and secondary fermentation are performed, finally temperature is dropped to 2-4 DEG C rapidly, acidity reaches to 81-degree T, and a finished product can be obtained. Bifidobacterium metabolic factors facilitate anti-aging of human bodies, improve human body immunization, and prevent cancer. The low-cholesterol bifidobacterium yoghurt is low in cholesterol milk and has the effect of regulation of blood pressure and blood fat. Important trophic factors capable of helping insulin to maintain normal glycometabolism are added, and the low-cholesterol bifidobacterium yoghurt has three effects and is novel functional healthy milk.