Abstract： Objective To explore the feasibility of using protamine-agarose gel to achieve heparin-free cardiopulmonary bypass （CPB）. Methods A total of 12 healthy adult dogs were chosen, the dogs were between 2-3 years old,either male or female, with their mean body weight of 23.3±3.7 kg （ranging from 20 to 28 kg）. All the dogs were randomly divided into two groups with 6 dogs in each group. In the heparinized group, conventional CPB technique was used; in the non-heparinized group, protamine-agarose gel column was used to absorb plasma clotting factors in CPB without use of heparin. At the beginning of CPB and 1 h, 2 h, 3 h after CPB, arterial blood samples were collected from dogs in both groups. The expression levels of tumor necrosis factor-α （TNF-α）, interleukin-6 （IL-6） and interleukin-8 （IL-8） were measured by enzyme-linked immunosorbent assay（ELISA）and compared. Results There was no thrombus formation in the membrane oxygenators during CPB by naked eye observation in both groups. Activated coagulation time （ACT） was always greater than 480 s during CPB. The vital signs of the dogs were all stable during CPB. At the beginning of CPB, there was no statistical difference in plasma concentrations of TNF-α, IL-6, IL-8 between the two groups. At 1 h, 2 h and 3 h after CPB, the expression levels of TNF-α and IL-8 of the non-heparinized group were significantly higher than those of the heparinized group （CPB 3 h TNF-α：156.48±16.65 ng/L vs. 115.87±15.63 ng/L, t=4.356, P=0.001；CPB 3 h IL-8：365.38±46.18 ng/L vs. 299.29±34.50 ng/L, t=2.808, P=0.019）. There was no statistical difference in the expression level of IL-6 between the two groups （P＞0.05）. Conclusion Using protamine-agarose gel to absorb plasma clotting factors is an effective technique to establish heparin-free CPB. But this method can induce significant systemic inflammatory response.