Immune checkpoint blockade (ICB) therapy represents a pivotal advancement in tumor immunotherapy by restoring the cytotoxic lymphocytes' anti-tumor activity through the modulation of immune checkpoint functions. Nevertheless, many patients experience suboptimal therapeutic outcomes, likely due to the immunosuppressive tumor microenvironment, drug resistance, and other factors. Single-cell RNA sequencing has assisted to precisely investigate the immune infiltration patterns before and after ICB treatment, enabling a high-resolution depiction of previously unrecognized functional interaction among immune checkpoints. This review addresses the heterogeneity between tumor microenvironments that respond to or resist ICB therapy, highlighting critical factors underlying the variation in immunotherapy efficacy and elucidating treatment failure. Furthermore, a comprehensive examination is provided of how specific ICBs modulate immune and tumor cells to achieve anti-tumor effects and generate treatment resistance, alongside a summary of emerging immune checkpoints identified as promising targets for cancer immunotherapy through single-cell RNA sequencing applications.