Abstract: In this study, the traditional cookie recipe was optimized to develop healthier, defatted soybean flour cookies. Low-gluten flour was used as the primary ingredient, with defatted soybean flour as a supplement. Butter and sucrose were partially replaced with grape seed oil and fructooligosaccharides, respectively. Single-factor and response surface methodology experiments were conducted. The final formulation was determined based on sensory evaluation, texture analysis, and impact on lipid metabolism in mice. The results indicated that the optimized formulation included 62.24 g low-gluten flour, 37.76 g defatted soybean flour, 42.66 g butter, 12.34 g grape seed oil, 19.61 g sucrose, 5.39 g fructooligosaccharides, and 35.00 g egg. The sensory score of the product was 90.40±4.69 higher than the traditional cookie's score of 83.08±4.85 (P<0.05). Although the cost of the optimized formulation was 1.40~1.50 times higher than that of the traditional formulation, it did not disrupt lipid metabolism in BALB/c mice. After 12 weeks of feeding, the defatted soybean flour cookie group mice exhibited significantly lower body weight, liver index, fat coefficient and serum liver damage indicators than those in the traditional cookie group (P<0.05). Total triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) of defatted soybean flour cookie group mice were significantly lower than the traditional cookie group while high density lipoprotein cholesterol (HDL-C) was significantly higher than the traditional cookie group (P<0.05). No significant lipid accumulation was observed in the liver tissues, similar to the observation in the maintenance diet group. These findings suggest that defatted soybean flour cookies are healthier and have promising market potential.