Safflower, an economic crop, is renowned for its flowers, which are widely used in medicines for treating cardiovascular and cerebrovascular diseases and in dyes for food and industry. The utility of safflower depends on its flavonoid glycosides. Therefore, the biosynthesis of safflower flavonoid glycosides has been a focus of attention, but the present mechanisms remain poorly understood. This study aims to identify functional genes associated with flavonoid glycoside biosynthesis in safflower through a comprehensive approach that integrates whole-genome screen and multi-omics correlation studies. CYP and UGT are two crucial genes families involved in flavonoid glycoside biosynthesis. We have screened 264 CYP genes and 140 UGT genes in the genome of safflower and conducted analyzes including phylogenetic relationships, conserved motifs, gene structures, -acting elements, and chromosome mapping, which provided extensive and comprehensive data on the CYP and UGT gene families. Integration of phenotype and metabolic data from safflower different tissues helped narrow down the screening by confirming that HSYA is synthesized only in flowers. Based on the gene expression patterns and phylogenetic analysis, was ultimately identified, which could catalyze the generation of glycosides using various flavonoid substrates and exhibited strong substrate affinity. Moreover, molecular docking studies elucidated CtOGT1's highly active intrinsic mechanism. In conclusion, this study effectively identified genes responsible for flavonoid glycoside biosynthesis in safflower through the integration of whole-genome screen and multi-omics analysis, established a comprehensive foundation of data, methodology, and experimental evidence for further elucidating the pathways of safflower flavonoid glycoside biosynthesis.