This investigation was carried out to produce biscuit consists of equal weight from different natural sources of dietary fiber. Different dietary fiber sources as defatted soybean, carrot powder and resistant starch were mixture and the chemical composition was determined in raw materials. The dietary fiber mixture were used at different levels 20, 30, 40 and 50% respectively, at preparing biscuit and the sensory evaluation was recorded and the control biscuit was made from wheat flour 72% extraction. The results showed that the defatted soybean had the highest content of fat 3.46% and protein 50.35%, whereas, the resistant starch had the highest content of total carbohydrates and total dietary fiber 98.69 and 39.56% moreover, the defatted soybean and carrot powder had the highest cellulose 14.11 and 12.01% and hemicelluloses 8.31 and 3.86%, respectively. The results of statistical analysis for sensory properties indicated that no significant difference was found between control sample and supplementation of the samples for all characteristics were evaluated of biscuits contained 20, 30 and 40% dietary fiber mixture The biological experimental for hypercholesterolemia and glycemia rat groups were fed on diets containing different dietary fiber sources mixture at different ratios. After four weeks of feeding, serum glucose, total lipids, total cholesterol and triglycerides were determined. High and low density lipoprotein- cholesterol in serum was determined and liver function enzyme activity (ALT) alanine and asparate (AST) transaminases were measured. At the end of experimental the results showed that increasing dietary fiber mixture in the diet had significantly decreased in levels of total cholesterol from 196.3 in control positive to 110.3 mg/dL in rats fed on 50% dietary fibers mixture. Also, triglyceride was lowered from 245.7 to 118.7 mg/dL, total lipid was decreased from 1.42 to 0.72 g/dLand glucose level was decreased from 169.3 to 120.6 mg/dL in blood serum of hypercholesterolemic rats. Meanwhile, the activities of aspartate and alanine transaminoferases were significantly reduced the actives in transaminoferases in the hypercholesterolemic rate group fed on high content of fiber mixture sources. Therefore, it may be recommended to use these sources of dietary fiber mixture in made from biscuits up to 40% and it has improved blood lipids patterns and liver functions.