To elucidate the distribution characteristics of astaxanthin in Pacific white shrimp (Litopenaeus vannamei), a robust method for the extraction and quantification of astaxanthin in shrimp tissues was developed. Methanol was identified as the optimal extraction solvent. A single-step extraction at a 1∶50 (w/v) solid-liquid ratio combined with a methanol-THF sequential dissolution approach was utilized. Through controlled feeding trials with graded astaxanthin supplementation (0, 40, 80, 120 mg/kg), we quantitatively analyzed the tissue-specific accumulation patterns, body color responses and associated enzymatic changes. The results indicated that astaxanthin significantly enhanced the body color of shrimp to a red-orange hue and was predominantly accumulated in the hepatopancreas, ventral nerve cord and epidermis. Additionally, astaxanthin supplementation effectively increased the total antioxidant capacity (Total antioxidant capacity, T-AOC) of the hepatopancreas, elevated the activities of superoxide dismutase (Superoxide dismutase, SOD), catalase (Catalase, CAT), alkaline phosphatase (Alkaline phosphatase, AKP) and glutathione peroxidase (Glutathione peroxidase, GSH-Px), and reduced the content of malondialdehyde (Malondialdehyde, MDA).These findings demonstrated that astaxanthin significantly enhances the antioxidant capacity and immune function of L. vannamei.