As critical splicing regulators, serine/arginine-rich splicing factors (SRSFs) play pivotal roles in carcinogenesis. As dysregulation of SRSFs may confer potential cancer risks, targeting SRSFs could provide important insights into cancer therapy. However, a global and comprehensive pattern to elaborate the molecular characteristics, mechanisms, and clinical links of SRSFs in a wide variety of human cancer is still lacking. In this study, a systematic analysis was conducted to reveal the molecular characteristics and clinical implications of SRSFs covering more than 10000 tumour samples of 33 human cancer types. We found that SRSFs experienced prevalent genomic alterations and expression perturbations in multiple cancer types. The DNA methylation, m6A modification, and miRNA regulation of SRSFs were all cancer context-dependent. Importantly, we found that SRSFs were strongly associated with cancer immunity, and were capable of predicting response to immunotherapy. And SRSFs had colossal potential for predicting survival in multiple cancer types, including those that have received immunotherapy. Moreover, we also found that SRSFs could indicate the drug sensitivity of targeted therapy and chemotherapy. Our research highlights the significance of SRSFs in cancer occurrence and development, and provides sufficient resources for understanding the biological characteristics of SRSFs, offering a new and unique perspective for developing cancer therapeutic strategies.