In order to optimize the extraction process and analysis of subunit component of glutelin from Euryale ferox seed, the effect of four variables including liquid/material ratio, temperature, time and sodium hydroxide concentration on the extraction yield of glutelin was investigated by alkali solution and acid isolation. The glutelin extraction model was fitted by means of Box-Behnken design. Moreover, the subunit component of glutelin from Euryale ferox seed was analyzed. The results indicated that the optimum conditions for glutelin in Euryale ferox were determined as 10.3 (V/m), 51 ℃, 2.2 h, 4.5 g/L for liquid/material ratio, temperature, time and sodium hydroxide concentration, respectively. Under these conditions, the extraction yield of glutelin was 32.45%, which was no significant difference with the theoretical value of 32.55% (P>0.05). Thus, the regression model has good predictive performance, and can be used to guide extraction of glutelin in Euryale ferox seed. And the molecular mass of about 15 kD is the major subunit component of glutelin from Euryale ferox seed, which accounts for 52.4%. It can be used to construct the Euryale ferox glutelin fingerprint and provide a theoretical basis.