The invention relates to non-fermentation type sour milk and a preparation method thereof. The non-fermentation type sour milk uses fresh cow milk as principal raw material. After being hydrolyzed by milk sugar, the cow milk is pasteurized and concentrated in vacuum to be 40 percent of the original volume. After the cow milk is cooled, probiotic powder is added. The invention has the core that transglutaminase is added at an optimal temperature; under the action of the transglutaminase, milk protein is connected together to form a protein network, thereby a uniform stable gelatin system is easily formed; delta-gluconolactone is then added, and the gelatin system and the delta-gluconolactone are heated at 35-40 DEG C in a water bath way for 2 to 3 hours so that the uniform, stable and exquisite non-fermentation type sour milk like a quark can be obtained. The invention has simple preparing process, no requirement for fermentation, and full utilization of resources. The non-fermentation type sour milk can be used as a base to develop various products, such as corn non-fermentation type sour milk for breakfast, jelly non-fermentation type sour milk, and the like, and can satisfy the requirements of consumers in nutrition, mouthfeel, texture, and the like.