The purpose of this research was to incorporate 0% to 30% chia seed flour into rice flour gluten‐free layer cake and evaluate its effect on nutritional qualities and physicochemical properties. The supplement of chia seed flour resulted in higher batter viscosity, hardness of gluten free layer cake, crude protein, fat, ash, α‐linolenic acid (increase up to 10.2% of total fatty acid), total phenolics content, and reducing power but the lower crust, crumb white index, cohesiveness, and resilience of gluten‐free layer cake was found. Substitution of rice flour with 10% prehydrated chia seed flour can achieve a higher center height and volume index of gluten‐free layer cakes. Higher carosine and angiotension I‐converting enzyme (ACE) inhibitory activity was observed in the supplement of 10% chia seed flour when compared to a rice flour layer cake. Gluten‐free layer cake with 10% prehydrated chia seeds flour had similar overall acceptability, texture, flavor, and odor scores except for lower appearance score to those of gluten‐free layer cake made with 100% rice flour and layer cake made with 100% wheat flour. Incorporation of 10% prehydrated chia seed flour results in more desirable volume index of gluten‐free layer cake and it is feasible for gluten‐free layer cake application. Gluten‐free layer cakes with improved quality characteristics and high nutritional value can be manufactured by the incorporation of chia seed flour.