Understanding how colorectal tumours interact with immune cells is critical for improving immunotherapy responses. In this study, we used air–liquid interface organoids derived from patient tumours and adjacent healthy colon tissues to reproduce the original tumour microenvironment in the laboratory. These organoids preserved the three-dimensional tissue structure, the native immune infiltrates, and the genetic mutations of the original samples more faithfully than conventional Matrigel-based cultures. When compared with Matrigel organoids co-cultured with blood immune cells, the air–liquid interface model more accurately reflected the immune and molecular features of each patient’s tissue. Notably, treatment with the immune checkpoint inhibitor nivolumab (anti–PD-1) led to a consistent expansion of regulatory T cells, recapitulating a mechanism of adaptive resistance. This approach offers a physiologically relevant platform to study colorectal cancer–immune interactions and to predict patient-specific responses to immunotherapy.