Fish protein isolate (FPI) nom Pangasius hypophthalmus by-products has many beneficial technological features that can be applied in food industry. Besides, it can bind calcium. This a~ility is regarded as one of its outstanding biological characteristics. This opens up prospects for the production of FPI to enhance calcium absorption. This study focuses on the optimization of the proteolytic conditions of the by-products of Pangasius hypophthalmus to obtain FPI with the highest calcium binding capacity. The enzymatic hydrolysis was performed using endoprotease (Alcalaza 2.4L) at different hydrolysis conditions. Optimization was done by response surface method. Results showed that the highest calcium binding capacity of 26.85 (mg Ca+2/g FPI) was obtained under the following hydrolysis conditions: pH 7.0; enzyme/substrate ratio (E/S) 0.15 percent w/v; hydrolysis temperature 55°C; hydrolysis time 120 minutes. Molecular weight analysis by HPLC shows that 82.88 percent FPI protein molecules are smaller than 3 kDa and 17.12 percent are from 3 to 7 kDa.