<p>Osteosarcoma, an aggressive bone cancer found most often in children and adolescents, remains difficult to treat, and little improvement in survival rate has been observed over recent decades. The tumor microenvironment (TME), especially the extracellular matrix (ECM), is a critical factor determining cancer progression and chemotherapy resistance, yet traditional 2D models generally fail to replicate its properties. Recent development of 3D-bioprinted tumor models has facilitated improved simulation of the complexity of the TME, but specific models involving bioinks tailored to osteosarcoma remain underdeveloped. Gelatin methacryloyl (GelMA) is a common bioink that can rapidly gel and contains Arg-Gly-Asp (RGD) sequences. However, it lacks collagen’s triple-helix structure that is essential for ECM–cell communication. Hyaluronic acid (HA) is a macromolecule that is aberrantly expressed in osteosarcoma by mechanisms that remain largely unexplored. In this study, we developed a composite bioink containing GelMA, collagen, and HA, and applied it to 3D bioprint an in vitro osteosarcoma model. We found that HA significantly enhanced osteosarcoma cell proliferation and chemoresistance, as well as the expression of epithelial–mesenchymal transition and cancer stem cell markers. Furthermore, we found that HA abundance was positively correlated with hypoxia and angiogenesis signaling pathways, and this occurred mainly via upregulation of hypoxia-inducible factor-1α (<i>HIF-1α</i>) and vascular endothelial growth factor A (<i>VEGFA</i>) expression, thereby contributing to increased chemoresistance. Overall, our study provides a protocol for building in vitro realistic 3D-bioprinted models for studying osteosarcoma, highlights the role of HA in osteosarcoma progression, and offers a platform for developing new chemotherapy treatments.</p>