The invention provides a bioactive substance composition, a serum-free medium comprising the composition and the uses thereof. The bioactive substance composition is used for serum-free medium and/or composition and the preparation thereof; The serum-free medium and/or composition can be used for primary culture and secondary culture of cells and/or tissues. The cells are selected from any one or more of tendon and/or ligament derived cells, chondrocytes, meniscus stem cells, mesenchymal stem cells, skeleton stem cells, and muscle stem cells. The tissue is the musculoskeletal system tissue. The bioactive substance composition and/or serum-free medium and/or the composition can be used to prepare drugs for tissue and/or organ injury treatment; The tissue or organ injury is selected from the tissue or organ injury of the musculoskeletal system.