Defatted roselle seed, a by-product of roselle calyx, was considered as a source of antioxidants. The feasibility of subcritical water extraction to release phenolic compounds in defatted roselle seed was investigated. The optimization of subcritical water extraction was obtained by a response surface methodology. The effect of different solvents used to obtain defatted roselle seed on the phenolic extraction was also studied. Defatted roselle seed contains valuable phenolic compounds that show high antioxidant activity. p-Coumaric acid and ferulic acid are the major phenolic compounds found in defatted roselle seed. Under optimum conditions, the subcritical water extract obtained has a phenolic content 12 times that of defatted roselle seed. The extract has a total phenolic content of 56.72 mg gallic acid equivalent per gram extract (analyzed by the Folin–Ciocalteu method), or 22.29 mg per gram extract (by HPLC analysis) and a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging activity of 84.0% at a concentration of 2.5 mg/mL.