An oral colon-targeted delivery system is described, which includes a bacterial flora sensitive layer which contains polysaccharides and covers the exterior of active ingredients, a pH sensitive layer which contains any polymer composition dissolved under the condition of pH≥;7 and covers the exterior of the bacterial flora sensitive layer. A preparation method and applications of the delivery system are also described. According to the oral colon-targeted delivery system combined with the pH sensitive and bacterial flora sensitive mechanisms, the selective dissolution and release of active ingredients in the colon site are effectively improved by using double-layer protection so that the therapeutic or diagnostic effect is enhanced, and the application prospect is broad.