Abstract: To establish a tumor-endothelial cell co-culture model with a spatially partitioned structure, this study employed a chitosan electrostatic adsorption coating strategy to surface-modify calcium alginate microspheres. Breast tumor cells (MCF-7) were encapsulated within the microspheres, while human umbilical vein endothelial cells (HUVEC) were seeded on the microsphere surface, mimicking the spatial relationship between tumor cell clusters and vascular endothelium in solid tumors. By systematically investigating the effects of varying chitosan concentrations (1、3、5、7 g/L) on microsphere particle size, surface charge, dimensional stability, and HUVEC adhesion, the optimal coating conditions were identified, and the proliferation behavior of tumor cells in the co-culture model was evaluated. Results indicated that microspheres modified with 5 g/L chitosan exhibited optimal particle size stability, a surface zeta potential of (9.00±0.57)mV, the highest HUVEC adhesion density with good spreading, and no significant cytotoxicity toward internal MCF-7 cells. In the co-culture model constructed using this optimized coating, HUVECs formed a continuous cell layer on the microsphere surface, connecting dispersed microspheres into tissue-like aggregates. MCF-7 cell activity in the co-culture group was enhanced compared to the sole culture group (reaching 115% by day 7). This model holds potential applications in studying tumor-endothelial cell interaction mechanisms, drug evaluation, and personalized medicine, providing a novel in vitro platform for simulating the tumor microenvironment.