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Intercellular communication is critical to the understanding of human health and disease progression. However, compared to traditional methods with inefficient analysis, microfluidic co-culture technologies developed for cell-cell communication research can reliably analyze crucial biological processes, such as cell signaling, and monitor dynamic intercellular interactions under reproducible physiological cell co-culture conditions. Moreover, microfluidic-based technologies can achieve precise spatial control of two cell types at the single-cell level with high throughput. Herein, this review focuses on recent advances in microfluidic-based 2D and 3D devices developed to confine two or more heterogeneous cells in the study of intercellular communication and decipher the advantages and limitations of these models in specific cellular research scenarios. This review will stimulate the development of more functionalized microfluidic platforms for biomedical research, inspiring broader interests across various disciplines to better comprehend cell-cell communication and other fields, such as tumor heterogeneity and drug screening., (© 2023 IOP Publishing Ltd.)