Abstract: To explore the genes related to anthocyanin accumulation and synthesis during the ripening process of strawberry fruit, qualitative and quantitative analysis of anthocyanin substances was conducted using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) on the fruits of four development stages of 'Benihoppe' strawberry: White (WF), initial ripening (IR), part ripening (PR), and full red fruits (FR). The expression level of structural genes in anthocyanin biosynthesis pathway in different development processes was determined to provide a theoretical basis for anthocyanin metabolism and color quality regulation of strawberry fruit. The results showed 24 anthocyanins and four proanthocyanidins in 'Benihoppe' strawberry. With the ripening of the fruit, the total anthocyanin and proanthocyanidin contents gradually increased and decreased, respectively. Pelargonidin and cyanidin were the main differential metabolites in different developmental stages. Among them, pelargonidin-3-O-glucoside, pelargonidin-3-O-rutinoside and cyanidin-3-O-glucoside were the main substances in anthocyanins, accounting for 79.7%, 14.1% and 3.56% of the total anthocyanins at the full maturity of 'Benihoppe' strawberry, respectively. Real-time PCR analysis showed that the transcription levels of the anthocyanin synthesis pathway genes FaPAL1, FaC4H, FaF3H, FaANS, FaUFGT, and FaMYB10 gradually increased with the fruit development. In summary, the anthocyanin content of 'Benihoppe' strawberry fruit gradually accumulates with fruit maturity, and the substances of anthocyanin differential metabolism include pelargonidin and cyanidin. The expression level of structural genes related to anthocyanin synthesis pathway affects the anthocyanin accumulation of fruit.