Tempeh is a traditional Indonesian food that is fermented soybeans with Rhizopus spp. and contains high-quality protein. In this study, tempeh and yeast were set as independent variables following the central composite design to optimize the nutritional quality and sensory characteristics of high-protein loaf bread added with tempeh. The optimal mixing ratio was calculated through the response surface methodology. The optimal mixing ratio of tempehadded loaf bread was 11.27 g of tempeh and 5.46 g of yeast. A comparative analysis of the quality characteristics between the tempeh-added loaf bread and the control group produced by the optimal recipe showed that tempehadded loaf bread contained higher protein content, lower carbohydrates and fats, and lower calories. Among amino acids, essential amino acids such as aspartic acid, threonine, glycine, alanine, lysine, and histidine were significantly higher in tempeh-added loaf bread. Among fatty acids, butyric acid, capric acid, myristic acid, palmitic acid, palmitoleic acid, and heptadecanoic acid were significantly higher in tempeh-added loaf bread, while lauric acid, linoleic acid, and linolenic acid were significantly lower. This study confirmed the suitability of tempeh as an ingredient for protein-enhanced loaf bread and the potential possibility of it being utilized in bakeries.