Colorectal cancer (CRC) is now the third most common cancer worldwide. However, the development cycle for anticancer drugs is lengthy and the failure rate is high, highlighting the urgent need for new tumor models for CRC-related research. The decellular matrix (dECM) offers numerous cell adhesion sites, proteoglycan and cytokines. Notably, porcine small intestine is rich in capillaries and lymphatic capillaries, which facilitates nutrient absorption. This study, we utilized dECM, along with methylacryloyl gelatin (GelMA), sodium alginate (SA) and nanoclay (NC) to create a hydrogel scaffold through 3D extrusion bioprinting. Human CRC cells (HCT8) were seeded onto the scaffold and their drug resistance was tested using 5-fluorouracil (5-FU). Our findings indicate that dECM enhances the hydrophilic properties, mechanical strength and biocompatibility of the scaffold. Furthermore, compared to traditional two-dimensional (2D) models, the three-dimensional (3D) scaffold supports the long-term growth of tumor spheres. After 2 days of 5-FU treatment, the cell survival rate reaches 88.06 ± 0.51 %. This suggests that our scaffold provides a promising alternative platform for in vitro research on cancer mechanisms, anti-cancer drug screening and new drug development.