SUMMARY Phloridzin has various functions, including antioxidant properties and the treatment of diabetes, and has long been used in pharmaceutical and physiological research. The glycosylation of phloretin is a key step in the biosynthesis of phloridzin. In this study, a genome‐wide association study (GWAS) based on phloridzin content was applied, and the key gene GhUGT88F3 for phloridzin‐specific biosynthesis was identified in cotton. A single‐base deletion in GhUGT88F3 in haplotype I caused a frameshift mutation, leading to premature translation termination and a significant reduction in phloridzin content. Molecular docking revealed important amino acid residues for GhUGT88F3's UDP‐glucose transfer activity. Additionally, the transcription factor GhMYB330 was found to positively regulate GhUGT88F3 expression through population transcriptome analysis and LUC experiment. Moreover, phloridzin content was significantly elevated in both GhUGT88F3 and GhMYB330 overexpression transgenic plants. This study expands the diversity of UDP‐glucosyltransferases in plants and offers a potential strategy for the sustainable production of bioactive compounds with therapeutic potential.