The involvement of Programmed death-ligand 1 (PD-L1) in tumor cells, with its receptor PD-1 on immune cells, can transmit an inhibitory signal to induce immune evasion. Although the immune checkpoint inhibitor PD-L1 antibody has achieved significant antitumor efficiency in clinical treatment, its wide clinical application still involves several side effects and individual selectivity. In our research, we intend to use the CRISPR interference (CRISPRi) system to suppress PD-L1 expression in tumor cells; we combine it with the epigenetic inhibitor azacytidine (AZA) for enhanced cancer immunotherapy. We first fabricate a reactive oxygen species (ROS)-responsive poly(β-amino ester) (PBAE)-S-AZA cationic polymer prodrug. The CRISPRi plasmid and PBAE-S-AZA prodrug are complexed to form the final nanoparticles via electrostatic interaction. The nanoparticles are uptaken in tumor cells with high efficiency, followed by the escape of the plasmid via the endosome/lysosome with the cooperation of PBAE. The release of the CRISPRi plasmid can lead to a suppression of PD-L1 in tumor cells and a relief of the immune checkpoint blockade. In the meantime, the epigenetic inhibitor AZA is also released from the nanoparticles due to the high intracellular ROS level to synergistically enhance the efficacy of immunotherapy. It is demonstrated that the nanoparticles can significantly increase the proportions of CD8⁺ T cells, CD4⁺ T cells and M1 macrophages in the tumor microenvironment while decreasing the proportion of regulatory T cells (Tregs) and M2 macrophages. Thus, the nanoparticles help to achieve the combination of genome editing, immunotherapy and epigenetic regulation effectively. This study provides a novel platform for promoting antitumor treatment and precision medicine.