Abstract: The objective of this study was to optimize the extraction process of total flavonoids from Aletris spicata (Thunb.) Franch. and explore its antioxidant, anti-inflammatory activity. With the yield of total flavonoids as an evaluation index, single factor tests were used to investigate the effects of ethanol concentration, material liquid ratio and the extraction time on the total flavonoids content of Aletris spicata (Thunb.) Franch. On this basis, the Box-Behnken method was used to optimize the extraction conditions. The antioxidant activity of total flavonoids from Aletris spicata (Thunb.) Franch. were evaluated by measuring DPPH free radical, ABTS⁺ free radical, hydroxyl free radical scavenging ability. The inflammatory model was established by lipopolysaccharide-induced RAW 264.7 macrophages to evaluate the anti-inflammatory activity of total flavonoids from Aletris spicata (Thunb.) Franch.. The results showed that optimum extraction process was ethanol concentration of 40%, material liquid ratio of 1:125 g/mL, the extraction time of 30 min. The average yield of total flavonoids from Aletris spicata (Thunb.) Franch. was 1.34%. The scavenging ability of total flavonoids on DPPH radical, ABTS⁺ radical and hydroxyl radical were positively correlated with the concentration, and the IC₅₀ values were 5.46, 21.69 and 56.01 µg/mL, respectively. The total flavonoids of Aletris spicata (Thunb.) Franch. could significantly inhibit the secretion of NO in LPS-induced RAW 264.7 macrophages within a certain concentration range, and the IC₅₀ value was 6.95 µg/mL. The extraction process of total flavonoids is stable and reliable, which can be used for the extraction of total flavonoids, and the total flavonoids from Aletris spicata (Thunb.) Franch. has certain antioxidant and anti-inflammatory activity.