The invention belongs to the technical field of food processing, and particularly relates to a method for preparing a yoghurt fermentation strain. The yoghurt fermentation strain includes lactobacillus casei, lactobacillus plantarum, lactococcus lactis cremoris and lactococcus lactis subsp lactis with an activity ratio of 1:2:1:1. First the four strains are activated respectively, so that the activity of the strains can be good, and the activity of prepared yoghourt is good, to achieve a good effect of lubricating the intestines; and the well-activated strains are mixed according to the activity requirements and then placed in yoghourt for fermentation, so that the prepared yoghourt is good in cheese flavor and antibacterial effect. First the strains are activated and then placed in the yoghurt for fermentation to prepare the yoghourt, so that the prepared yoghourt is solid in a forming state; the four stains are mixed according to a certain proportion, the strain ratio is reasonable,the prepared yoghourt is good in taste and solid in the forming state, and the yoghourt is good in cheese flavor and has more outstanding antibacterial effects and intestine-lubricating effects than conventional commercial strains.