The Human Genome Project marked a milestone in scientific exploration, unraveling the genetic blueprint of humanity. However, expectations of direct gene–disease associations gave way to realizing the complexity of genetic interactions, especially in polygenic diseases. This review explores the legacy of the HGP and subsequent advancements in genomic technologies, particularly next-generation sequencing, which have enabled more profound insights into the non-coding genome's role in gene regulation. While initially dismissed as “junk” DNA, non-coding regions are now officially approved as critical gene expression and genome organization regulators. Through integrative genomics approaches and advanced computational methods, researchers have unveiled the intricate network of enhancers, promoters, and chromatin modifications orchestrating gene expression. High-throughput sequencing techniques and functional assays have identified non-coding variants associated with numerous diseases, challenging the conventional focus on coding sequences in genomic studies. By elucidating the regulatory mechanisms governing gene expression, researchers can advance precision medicine approaches and develop novel diagnostic tools. As genomic research continues to evolve, a vast landscape is waiting to be explored, promising transformative insights into human health and disease. This review provides a comprehensive overview of the non-coding genome's role in gene regulation and its implications for understanding complex diseases and developing targeted therapeutic interventions.