The present invention relates to equine amniotic fluid-derived multipotent stem cells (eAF-MSCs) and a preparation method thereof. More particularly, the present invention relates to equine amniotic fluid-derived multipotent stem cells which exhibit all negative immunological characteristics with respect to the human markers, CD19, CD20, CD28, CD31, CD34, CD38, CD41a, CD62L, CD62P and CD200, and exhibit all positive immunological characteristics with respect to the human markers, CD44, CD90 and CD105, and have the ability to differentiate into ectoderm, mesoderm or endoderm-derived cells.The present invention confirmed that equine amniotic fluid can be a source of equine adult multipotent stem cell supply, and the equine amniotic fluid-derived multipotent stem cells exhibit excellent self-renewal ability and differentiation capability. Therefore, the cells can be used as an active ingredient for cell therapy of horses, which requires a large amount of cells. In particular, the stem cells of the present invention having excellent self-renewal ability and differentiation capability are useful for the treatment of bone, tendon, or muscle injuries and bone loss diseases of racehorses.