Herein, we developed an integrated microfluidic cell assembly chip (MCA-chip) to engineer controlled cell assemblies for cell–cell interaction profiling at the single-cell level. The MCA-chip can: (1) Efficiently manipulate single cells; (2) construct multicellular combinations by precise on-demand cell assembly; (3) generate disconnected assembly chambers to prevent crosstalk among cell assemblies. We employed the MCA-chip to profile the immune response of single macrophages in paracrine signaling and transcriptomic changes in tumor microenvironment to provide insights into the interactions between immune cells and tumor cells.