To optimize the determination method for the total flavonoids in Lycopus lucidus, this study compared the absorption spectra of 6 reference substances and the samples in the three color reaction systems of NaNO2-Al(NO3)3-NaOH, AlCl3-Kac, and triethylamine. The response surface-Box-Behnken design optimization was carried out to optimize the influence of the color reagent dosage on the measurement results of total flavonoids in color reaction system of NaNO2-Al(NO3)3-NaOH. The uncertainty evaluation of the preparation of the test solution, the preparation of the reference solution, the color reaction system, and the standard curve fitting was carried out. The combined uncertainty also was calculated, and the expanded uncertainty and the confidence level of the measurement result were given. Through adaptability analysis, NaNO2-Al(NO3)3-NaOH color reaction system was finally selected in the experiment, and the optimal color reaction conditions were as follows: 0.9 mL of 50 g/L sodium nitrite, 0.6 mL of 100 g/L aluminum nitrate, 4.8 mL of 1 moL/L hydrogen sodium oxide, and color reaction temperature was 42 ℃. The content of total flavonoids was measured at (91.052±2.75) mg under optimal conditions, which provided a new reference method for the determination of total flavonoids.
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