The invention relates to a docetaxel (DTX) encapsulated OHAS-CTS (oligosaccharides of hyaluronic acid-chitosan) microsphere, a preparation method and application thereof. The invention adopts an EDC/NHS method to synthesize an OHAS-CTS coupled product, the preparation process is simple and the cost is low. Emulsification-crosslinking process is employed to prepare the microsphere the preparation process is simple, the microsphere surface is smooth, the microsphere with good morphology, no adhesion and uniformly distributed particle size can be obtained, and batch production of microsphere preparation can be realized. Drug loading ratio, encapsulation rate, drug release, related cell experiments and the like are carried out on the docetaxel encapsulated microsphere, and lay the foundation for clinical application. The prepared DTX-OHAS-CTS microsphere has good tumor cell inhibiting effect, and can be used as a targeting drug carrier for treating tumors.