The invention discloses a preparation method of bi-crosslinking stem cell sphere hydrogel for osteoarthritis, and belongs to the field of biological material preparation. The hydrogel can be used as a viscous substitute for synovial fluid after a cartilage operation, and is used for treating osteoarthritis, subchondral injury, osteoporosis, synovitis, tenosynovitis, tendinitis, tendon degeneration and interstitial cystitis. The hydrogel is injectable bi-crosslinking hydrogel and is prepared by modifying hyaluronic acid from an articular cavity matrix and chondroitin sulfate through hydroxyl groups and activating bi-crosslinking through EDC (Ethylene Dichloride). The bi-crosslinking stem cell sphere hydrogel obtained through reaction has good mechanical performance and controllable degradation rate, facilitates survival of 3D cell spheres, can moisturize, lubricate and slowly release biological factors for a long time, has high drug loading rate and light responsiveness, and has potential application value in the fields of injectable drug slow release carriers, minimally invasive therapy and the like.