To investigate the affinity constants of heparin with high mobility group protein 1(HMGB1) and HMGB1 with the receptor of advanced glycation end products (RAGE) and to analyze the impact of heparin on the affinity of HMGB1 and RAGE, the standard BIAcore amine coupling chemistry protocol using EDC and NHS was employed for immobilizing. Surface plasmon resonance biosensor technology was used to detect the affinity constants of heparin/HMGB1, HMGB1/RAGE and heparin/RAGE. Binding analysis was used to investigate the impact of heparin on the affinity of HMGB1 and RAGE. After the immobilization, 9 000 and 5 000 RU rise of HMGB1 and RAGE respectively were obtained. These meant that the immobilized values of HMGB1 and RAGE were about 9 and 5 ng·mm-2 respectively. The kinetic constants were ka=1.78×105 L·mol-1·s-1, kd=8.02×10-4 s-1, and the affinity constants were KA=2.22×108 L·mol-1, the equilibrium dissociation constant KD=4.5×10-9 mol·L-1 for heparin and HMGB1; while the kinetic constants were ka=1.85×103 L·mol-1·s-1, kd=1.81×10-4 s-1, KA=1.02×107 L·mol-1, KD=9.77×10-8 mol·L-1 for HMGB1 and RAGE; there was very low affinity of heparin with RAGE. The highest concentration of 10 000 u·L-1 of heparin in this experiment did not reach the saturation with HMGB1. After 50 mg·L-1 of HMGB1 was mixed with heparin of 50, 100, 1 000, 10 000 u·L-1, the combining amount of HMGB1 and RAGE declined from 100 to 50 RU. But there were no significant differences between different concentrations of heparin. It was concluded that heparin can combine with HMGB1 and affect the affinity of HMGB1/RAGE. In addition, this impact was not in a dose-dependent manner.