Abstract: This study was aimed to extract the total flavonoids from the flowers of Abelmoschus manihot (L.) Medicus, clarify the composition of the individual flavonoid compounds as well as its ameliorative effect on hyperuricemia in mice. The dried flower of Abelmoschus manihot (L.) Medicus was used as raw materials for this study. Both single-factor and response surface approaches were performed to optimize the parameters for the isolation of total flavonoid from Abelmoschus manihot (L.) Medicus. The molecular composition of Abelmoschus manihot (L.) Medicus extract was analyzed using the targeted metabolomics approach. Furthermore, an animal model of hyperuricemia was established to elucidate the hypouricemic activity of total flavonoid. The maximum content of total flavonoid could reach 19.73±0.11 mg/g when the extraction lasting for 35 min under the conditions set at a liquid/material ratio 25:1 mL/g, ultrasonic power 250 W and microwave power 190 W. The targeted metabolomics data revealed that the dominant flavonoid compounds were myricetin, phenylalanine, rutin, quercetin 3-glucoside, quercetin, etc. Compared to the level of uric acid (124.95±26.22 μmol/L) in the hyperuricemic mice, the administration with low- and high-dose of total flavonoid extract significantly decreased the uric acid levels, being 52.62±2.76 μmol/L and 43.80±9.94 μmol/L, respectively. Our findings indicated that total flavonoid extract could exert hypouricemic effects by alleviating kidney and liver damage in mice, lowering levels of interleukin-1β, interleukin-6, tumor necrosis factor, creatinine and urinary nitrogen in the serum, malondialdehyde content in the kidney, and xanthine oxidase activity in the liver, while enhancing the activity of superoxide dismutase and catalase in the kidney. These findings are expected to provide theoretical guidance and data support for the value-added utilization of Abelmoschus manihot (L.) Medicus and also for the development of novel hypouricemic products.