Abstract: Roselle (Hibiscus sabdariffa L.) seed, commonly discarded as waste, is known as a good source of protein. Roselle seed protein is mostly composed of albumin, globulin and glutelin, covering 82.31% of total nitrogen in roselle seed, and soluble in water, salt and dilute alkalizes solution, respectively. Roselle seed protein was reported to be highly soluble (90-95%) at pH 9. However, oligosaccharides and phytic acids may extensively bind to protein and hence could significantly interfere protein isolation. Based on other studies, α-amylase (E.C. 3.2.1.1) can be used to release the bound protein into solution and it has higher effect than other carbohydrates on the extraction of protein. Thus, it is advantageous to isolate protein from defatted roselle seed by using water and salt solution at pH 9. Two-steps protein extraction using deionized water and salt solution at pH 9 resulted in 52.24% protein yield. This study found that the addition of α-amylase pretreatment (1 800 units/g DRSF for 6 h) resulted in 72.18% protein yield. Molecular weights of roselle protein, which has never been reported, range from slightly below 22 kDa to 95 kDa. Methionine is the main limiting amino acid. Roselle protein concentrate obtained in this study is rich in glutamic acid (28.78%), arginine (10.21%), aspartic acid (10.11%) and leucine (6.36%).