Objective To construct a bionic microfluidic multicellular chip model of ovarian cancer, which can be used to simulate the tumor microenvironment in vitro and systematically evaluate the efficacy and related pathological changes of chimeric antigen receptor T cell (CAR-T) therapy. Methods A total of 106 patients with epithelial ovarian cancer (EOC) were prospectively collected from surgical specimens, which were integrated with cancer-associated fibroblasts (CAFs) and human umbilical vein endothelial cells (HUVECs) to construct a three-dimensional multicellular tumor sphere model in microfluidic microarray. Moreover, CAR-T cells targeting mesothelin (MSLN) were injected into the microfluidic chip for dynamic perfusion co-culture. According to the different intervention methods, they were divided into: the MSLN-CAR-T group (n＝10) and the ordinary T cell group (n＝10). After 7 days of cultivation, the multicellular spheroids within the chip were collected. Pathological staining was employed to observe the pathological structure of the model, and the heterogeneity of target antigen expression in the model was evaluated. The survival rate of tumor cells was detected using cell viability/death staining. Immunofluorescence double-labeling staining was used to detect the infiltration ability of CART cells. The activation status of endothelial cells was detected by immunofluorescence double-labeling staining. The enzyme-linked immunosorbent assay (ELISA) was used to separately detect the concentrations of interleukin (IL)-6, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) in the perfusate. Results A microarray model of ovarian cancer covering multiple pathological subtypes was successfully constructed. After 72 hours of co-culture, the survival rate of tumor cells in MSLN-CAR-T group was significantly lower than that in the ordinary T cell group (P＜0.001), and the infiltration depth of CAR-T cells was significantly greater than that in the ordinary T cell group (P＜0.001). The concentrations of IL-6, TNF- α and IFN- γ all showed an increasing trend over time (all P＜0.05). The expression intensity of intercellular adhesion molecule-1 (ICAM-1) around the blood vessels in the MSLN-CAR-T group significantly increased as the distance from the tumor sphere decreased (P＜0.05). Conclusion The chip model established in this study can effectively simulate the tumor microenvironment in vivo, thus providing a high-throughput, reproducible and accurate platform for preclinical evaluation of CAR-T therapy.