Alternative splicing (AS) is a critical mechanism in transcriptional regulation, generating transcript diversity and modifying protein structure and function. AS-related RNA-binding proteins (RBPs) play a crucial role in regulating AS events, influencing protein structure, function, interaction, and localization. These processes are essential for tissue and organ development and are associated with various diseases, particularly cancer. Abnormal AS and RBPs can lead to altered gene splicing patterns and changes or loss of protein functionality, promoting tumor development. AS and RBPs are also linked to neurodegenerative and autoimmune diseases. The study of AS across different tissues is valuable for understanding gene expression complexity and advancing precision medicine. Recent advancements in high-throughput sequencing and CRISPR/Cas9 technology have enhanced our understanding of AS and AS-related RBPs, revealing their roles in disease progression and providing new insights into therapeutic strategies. The review covers the historical milestones, mechanisms of AS, the structure and function of RBPs, and their regulatory roles in physiological and pathological processes, with a focus on tumors and neurodegenerative diseases. It also discusses the latest methods for studying AS and RBPs, including computational approaches and high-throughput techniques. The physiological functions of AS-related RBPs in various tissues, such as the reproductive, neural, digestive, and immune systems, are highlighted, emphasizing their roles in tissue development and disease.Alternative splicing (AS) is a critical mechanism in transcriptional regulation, generating transcript diversity and modifying protein structure and function. AS-related RNA-binding proteins (RBPs) play a crucial role in regulating AS events, influencing protein structure, function, interaction, and localization. These processes are essential for tissue and organ development and are associated with various diseases, particularly cancer. Abnormal AS and RBPs can lead to altered gene splicing patterns and changes or loss of protein functionality, promoting tumor development. AS and RBPs are also linked to neurodegenerative and autoimmune diseases. The study of AS across different tissues is valuable for understanding gene expression complexity and advancing precision medicine. Recent advancements in high-throughput sequencing and CRISPR/Cas9 technology have enhanced our understanding of AS and AS-related RBPs, revealing their roles in disease progression and providing new insights into therapeutic strategies. The review covers the historical milestones, mechanisms of AS, the structure and function of RBPs, and their regulatory roles in physiological and pathological processes, with a focus on tumors and neurodegenerative diseases. It also discusses the latest methods for studying AS and RBPs, including computational approaches and high-throughput techniques. The physiological functions of AS-related RBPs in various tissues, such as the reproductive, neural, digestive, and immune systems, are highlighted, emphasizing their roles in tissue development and disease.