The aim of this study was to evaluate the influence of incorporation of xanthan gum and flaxseed flour on physicochemical, antioxidant properties and qualities of gluten-free layer cake replaced rice flour up to 60%. The incorporation of flaxseed flour increased crude protein, crude fat, ash and α-linolenic acid (increase up to 14.67% of total fatty acid with the replacement of 60% rice flour). Replacement of rice flour with flaxseed flour can mitigate the rate of hardness increase in gluten-free layer cakes. Significantly higher total phenolics content (114% increase), 1,1-diphenyl-2-picrylhydrazyl hydrate (DPPH) radical scavenging activity (124% increase) and reducing power (213% increase) was observed in the addition of flaxseed flour comparing to rice flour layer cake. 60% Flaxseed flour supplemented cake resulted in higher batter viscosity but lower crust and crumb white index, batter density, center height, volume index and symmetry index of gluten-free layer cake. Sensory evaluation showed rice flour gluten-free layer cake with 0.8% xanthan gum addition had higher aroma, texture, flavor and overall acceptability followed by 40% flaxseed flour replaced cake. Incorporation of 40% flaxseed flour results in better characteristics gluten-free layer cakes and it is feasible for gluten-free cake application.