Cervical cancer, primarily squamous cell carcinoma (CSCC), is the most common gynecological malignancy. Organoids can mimic tumor development in vitro, but current Matrigel inaccurately replicates the tissue-specific microenvironment, compromising the accurate representation of tumor heterogeneity. This study used uterine cervix extracellular matrix (UCEM) hydrogels derived from para-cancerous cervical tissues to create a more accurate microenvironment for CSCC organoids. UCEM preserved the structural and functional components of cervical ECM, including collagen, fibronectin, and laminin, as well as signaling pathways and secreted proteins associated with carcinogenesis. UCEM-organoids exhibited greater similarity to native CSCC tissues and reflected tumor heterogeneity by exhibiting CSCC-associated signaling pathways. UCEM-organoids also developed chemotherapy resistance, suggesting that UCEM provides a more accurate representation of the tumor microenvironment. The study demonstrated that UCEM supports the formation and growth of CSCC organoids, with higher success rates compared to Matrigel. UCEM-organoids exhibited similar transcriptomic characteristics to native CSCC tissues, with higher expression of oncogenes and signaling pathways linked to CSCC. UCEM-organoids also showed greater resistance to chemotherapy drugs, indicating that UCEM provides a more accurate model for drug development and precision medicine. The study highlights the importance of tissue-specific ECM microenvironments for organoid culture and suggests that UCEM could be a valuable tool for cancer research and treatment.Cervical cancer, primarily squamous cell carcinoma (CSCC), is the most common gynecological malignancy. Organoids can mimic tumor development in vitro, but current Matrigel inaccurately replicates the tissue-specific microenvironment, compromising the accurate representation of tumor heterogeneity. This study used uterine cervix extracellular matrix (UCEM) hydrogels derived from para-cancerous cervical tissues to create a more accurate microenvironment for CSCC organoids. UCEM preserved the structural and functional components of cervical ECM, including collagen, fibronectin, and laminin, as well as signaling pathways and secreted proteins associated with carcinogenesis. UCEM-organoids exhibited greater similarity to native CSCC tissues and reflected tumor heterogeneity by exhibiting CSCC-associated signaling pathways. UCEM-organoids also developed chemotherapy resistance, suggesting that UCEM provides a more accurate representation of the tumor microenvironment. The study demonstrated that UCEM supports the formation and growth of CSCC organoids, with higher success rates compared to Matrigel. UCEM-organoids exhibited similar transcriptomic characteristics to native CSCC tissues, with higher expression of oncogenes and signaling pathways linked to CSCC. UCEM-organoids also showed greater resistance to chemotherapy drugs, indicating that UCEM provides a more accurate model for drug development and precision medicine. The study highlights the importance of tissue-specific ECM microenvironments for organoid culture and suggests that UCEM could be a valuable tool for cancer research and treatment.