Abstract: To efficiently develop and utilize bamboo shoot by-products, optimize extraction process for total flavonoids from bamboo shoot, and enhance process data's rigor, an uncertainty assessment was conducted for total flavonoid content determination．Based on single-factor experiments, response surface methodology was employed to design and optimize extraction process for total flavonoids from bamboo shoot heads．Uncertainty sources have been analyzed, and to evaluate of total flavonoid measurement results' uncertainty has been evaluated．Optimal extraction process conditions obtained through response surface methodology were as follows: ethanol volume fraction of 60%, extraction temperature of 50 °C, extraction time of 70 minutes, and liquid-solid ratio of 30: 1 mL/g．Under these conditions, actual extraction rate of total flavonoids from bamboo shoot heads reached 0.435 mg/g．Uncertainty assessment results have indicated that uncertainty sources in total flavonoids determination for bamboo shoots heads primarily originated from standard solution preparation and standard curve fitting, with a reported value of （0.4353 ± 0.0580） mg/g．Extraction process for total flavonoids in bamboo shoot optimized by response surface methodology has been demonstrated to be reliable．Uncertainty assessment has quantified reliability of results, further enhancing process data's rigor．