Abstract Background Immune checkpoint blockades (ICBs) have emerged as a promising treatment for cancer. Recently, tumour mutational burden (TMB) and neoantigen load (NAL) have been proposed to be potential biomarkers to predict the efficacy of ICB; however, they were limited by difficulties in defining the cut-off values and inconsistent detection platforms. Therefore, it is critical to identify more effective predictive biomarkers for screening patients who will potentially benefit from immunotherapy. In this study, we aimed to identify comutated signaling pathways to predict the clinical outcomes of immunotherapy. Methods Here, we comprehensively analysed the signaling pathway mutation status of 9763 samples across 33 different cancer types from The Cancer Genome Atlas (TCGA) by mapping the somatic mutations to the pathways. We then explored the comutated pathways that were associated with increased TMB and NAL by using receiver operating characteristic (ROC) curve analysis and multiple linear regressions. Results Our results revealed that comutation of the Spliceosome (Sp) pathway and Hedgehog (He) signaling pathway (defined as SpHe-comut⁺) could be used as a predictor of increased TMB and NAL and was associated with increased levels of immune-related signatures. In seven independent immunotherapy cohorts, we validated that SpHe-comut⁺ patients exhibited a longer overall survival (OS) or progression-free survival (PFS) and a higher objective response rate (ORR) than SpHe-comut⁻ patients. Moreover, a combination of SpHe-comut status with PD-L1 expression further improved the predictive value for ICB therapy. Conclusion Overall, SpHe-comut⁺ was demonstrated to be an effective predictor of immunotherapeutic benefit in seven independent immunotherapy cohorts and may serve as a potential and convenient biomarker for the clinical application of ICB therapy.