The engagement of programmed death-ligand 1 (PD-L1) on 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 significant antitumor efficiency in clinical treatment, its widespread clinical application still faces several side effects and individual selectivity. In our research, we use the CRISPR interference (CRISPRi) system to suppress PD-L1 expression in tumor cells and combine it with 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. CRISPRi plasmid and PBAE-S-AZA prodrug are complexed to form the final nanoparticles via electrostatic interaction. The nanoparticles exhibit high efficiency in uptake by tumor cells, followed by the endosome/lysosome escape of plasmid with the cooperation of PBAE. The release of CRISPRi plasmid can lead to the suppression of PD-L1 in tumor cells and relief of immune checkpoint blockade. Meanwhile, the epigenetic inhibitor AZA is also released from the nanoparticles due to the high intracellular ROS level to enhance the efficacy of immunotherapy synergically. It was demonstrated that the ROS-responsive nanoparticles could significantly increase the proportion of CD8⁺ T cells, CD4⁺ T cells, and M1 macrophages in a tumor microenvironment, while decreasing the proportions of regulatory T cells (Treg) and M2 macrophages. Thus, the nanoparticles help to realize the combination of genome editing, immunotherapy, and epigenetic regulation effectively. Our method provides a novel platform for promoting antitumor treatment and precision medicine.