This study investigated the physicochemical properties and acceptability of yellow layer cake prepared using various levels of wet-milled rice flour (RF). RF was used to substitute 0% (control group), 15% (RF-15 group), 30% (RF-30 group) and 45% (RF-45 group) of wheat flour (WF) to manufacture yellow layer cakes. The substitution of WF with RF showed no significant effect on the pH but decreased the specific gravity and viscosity of the batter. Baking loss rates ranged from 4.65% to 5.03%, showing no significant difference among samples. In the color measurement, the experimental groups showed higher redness (a) and yellowness (b) than the control group (p<0.05). The experimental groups showed lower gumminess and chewiness than the control group. According to the results of intensity measurement of sensory properties, the RF-30 and RF-45 groups showed stronger intensities for darkness, sweetness, moistness and softness than the control group. The acceptance test found that the RF-45 group had significantly higher or similar results for all acceptance attributes than the control group. These results indicate that RF addition as a replacement of WF at a ratio of 45% is optimal for producing yellow layer cakes.