Objective This study aimed to construct a cell co-culture chip based on droplet microfluidic to investigate the influence of multicellular interactions in complex microenvironments on the sensitivity of anti-tumor drugs. Methods We constructed a droplet microfluidic chip consisting of 16 co-culture units, with each unit containing 4 microwells for holding cell droplets, enabling the co-culture of 4 types of cells. To evaluate whether the co-culture of multiple cell types can be achieved in the droplet co-culture system, as well as to observe and analyze the interactions between different cell types, we investigated the interaction between microenvironmental cells and tumor cells through cell co-culture experiments. To construct a stable co-culture system capable of evaluating drug sensitivity, we conducted diffusion experiments with blue ink and model drugs to investigate the ability of drugs to diffuse in the chip and be taken up by cells. To investigate the effect of the complex microenvironment on cellular drug sensitivity, we carried out cell co-culture experiments combined with drug treatment to explore the changes in the drug sensitivity of tumor cells in the presence of microenvironmental cells. To explore the reasons for drug resistance in tumor cells under co-culture conditions, we detected DNA double-strand break markers using immunofluorescence. Results Experiments on cell culture within droplets showed that cells in each droplet exhibited good proliferation ability and consistent cell status, laying a foundation for the co-culture of multiple cell types. Cell co-culture experiments showed that compared with the mono-culture group, the numbers of LoVo cells, HUVECs, and macrophages in the co-culture group increased significantly. This confirms that there are obvious interactions between cancer-associated fibroblasts (CAFs), endothelial cells (HUVECs), macrophages, and tumor cells in the microenvironment, which promotes cell proliferation in the co-culture chip. Experiments on blue ink diffusion showed that drugs could diffuse uniformly and effectively into wells in different directions within the chip. Experiments on the diffusion of doxorubicin (a model drug) demonstrated that identical cells in different wells exhibited consistent drug uptake capacity for the drug. Additionally, cell co-culture experiments combined with drug treatment revealed that at a working concentration of 80 μM, the survival rate of LoVo cells cultured alone was only 25%, whereas it reached 96% under co-culture conditions. At a working concentration of 160 μM, the survival rate of LoVo cells cultured alone was merely 2%, while that under co-culture conditions was 50%. These results indicate that the complex microenvironment composed of CAFs, HUVECs, and macrophages significantly reduces the drug sensitivity of LoVo cells to oxaliplatin through intercellular interactions. The immunofluorescence results showed that the expression level of γ H2AX in LoVo cells decreased under co-culture conditions. Conclusion Our study achieved co-culture of the main constituent cells of the tumor microenvironment and analysis of their drug sensitivity in a droplet microfluidic chip for the first time. The research found that crosstalk between different microenvironmental cells strongly affects the drug sensitivity of tumor cells to oxaliplatin, suggesting that targeting the interactions between tumor microenvironmental cells is an effective strategy to improve the efficacy of tumor therapy. Our study provides new methods and approaches for the efficacy evaluation of anti-tumor drugs and the screening of new drugs. In addition, the open structural design of this study can be combined with various omics technologies to analyze the molecular characteristics of cells under co-culture and drug treatment conditions. This is expected to provide new methods and experimental evidence for elucidating the mechanisms of drug action and identifying novel drug targets in the context of the microenvironment.