Next-generation sequencing (NGS) transforms genomics research by analyzing millions of DNA fragments concurrently, revealing genome structures, genetic variations, and gene activities in detail. Recent progressions prioritize faster, more precise sequencing, cost efficiency, and enhanced data interpretation, heralding fresh insights into genomics and disease understanding for personalized healthcare. This review provides a comprehensive exploration of NGS technology, delineating its profound influence across various research realms, such as clinical genomics, cancer, infectious diseases, and microbiome investigations. Through NGS, transformative opportunities emerge, reshaping the landscape of genomic exploration and healthcare advancements for future breakthroughs.