The invention provides a preparation method of a human body lubricant on the basis of a probiotic culture substance. In the technical scheme, probiotics, having beneficial effects on human body, are added to the human body lubricant with uniform mixing under a sterile condition, so that the probiotics are uniformly and fully distributed in vagina of female during use, thereby achieving lubrication effect and recovering microflora balance in vagina. With the flora advantage of the probiotics, colonization rick of other microorganisms is reduced. The human body lubricant effectively prevents and treats bacterial vaginitis, colpitis mycotica, trichomonas vaginitis, and the like gynecological diseases. In addition, in a manner of probiotics entering vagina, the microflora balance is regulated, so that the human body lubricant is convenient to use and has both treatment and prevention functions. A woman can fully enjoy sex and meanwhile is free from bacterial infection. The human body lubricant also can help treatment of different diseases caused by microflora disturbance in vagina, has good treatment effect, is free of relapse after healing, and has excellent promotion prospect.