Flavonoids, a variety of plant secondary metabolites, are known for their diverse biological activities. Isoflavones are a subgroup of flavonoids that have gained attention for their potential health benefits. Puerarin is one of the bioactive isoflavones found in the Kudzu root and Pueraria genus, which is widely used in alternative Chinese medicine, and has been found to be effective in treating chronic conditions like cardiovascular diseases, liver diseases, gastric diseases, respiratory diseases, diabetes, Alzheimer’s disease, and cancer. Puerarin has been extensively researched and used in both scientific and clinical studies over the past few years. The purpose of this review is to provide an up-to-date exploration of puerarin biosynthesis, the most common extraction methods, analytical techniques, and biological effects, which have the potential to provide a new perspective for medical and pharmaceutical research and development.