Abstract: To fully utilize chicory resources and explore the value of chicory bitterness in by-products from chicory powder processing, this study based on single-factor experiments, used ultrasonic technology combined with response surface methodology to extract chicory root bitters and optimize the parameters, aiming to obtain the optimal extraction parameters. Meanwhile, liquid chromatography-mass spectrometry (LC-MS) was used to analyze the components of chicory bitterness. Finally, the in vitro antioxidant activity of chicory bitters was investigated. The results showed that under the conditions of 80% ethanol concentration, ultrasonic temperature of 35 ℃, ultrasonic time of 24 min, ultrasonic power of 450 W, and liquid-to-material ratio of 33:1 mL/g, the yield of chicory bitterness was 1.18%±0.015%. Liquid chromatography-mass spectrometry analysis revealed seven main compounds in chicory bitterness, including andrographidine, quinpirole A, quinpirole B, chlorogenic acid, lactucinic acid, lactucin, and lactucopicrin. The partial in vitro antioxidant activity analysis of chicory bitterness indicated IC₅₀ values of 0.271, 0.0734, and 0.130 mg/mL for DPPH·, ABTS⁺·, and hydroxyl radicals, respectively, showing a concentration-dependent clearance ability. Additionally, at a concentration of 1.0 mg/mL, chicory bitterness exhibited a total reducing power of 1.108. This study provides a theoretical and practical basis for the utilization of chicory bitterness.