Abstract: The study aims to explore and optimize the extraction of carotenoids from pepper fruit and highperformance liquid chromatography (HPLC) detection system. Mature pepper fruits were used as materials, by using single factor experiment and L9(33) orthogonal test, the main five factors including extraction solvent, the ratio of solid to liquid, extraction temperature, time and pretreatment method to the raw materials were discussed. And then the types of mobile phase, ratio and flow fate were also optimized. The results showed that by using dichloromethane-ethyl alcohol (V: V = 1:1) as extraction solvent, the material to liquid ratio of 1:18, extraction temperature and time of 50℃ and 60 min, the yield of carotenoids was the highest. Using L600-2 HPLC, the main components of carotenoids in pepper fruit could be identified by the optimized chromatographic condition such as: methanol and acetonitrile as mobile phase A (V: V =55:45), 100% methyl tertiary butyl ether as mobile phase B and VA: VB=90: 10, 1.0 mL/min of flow rate and 25℃ of column temperature, 20 μL of sample quantity, respectively. This optimized carotenoids extraction method and HPLC determination method are easily operated, have high productivity and good repeatability, and can provide a reference for the extraction and determination of carotenoids from pepper fruit in the future.