The interaction of acridine orange (AO) with heparin was studied by voltammetric method. In Britton-Robinson (B-R) buffer solution (pH 2.3), AO showed a sensitive linear sweep voltammetric reductive peak at -0.94 V (vs. SCE). The addition of heparin into the AO solution caused a decrease in the reductive peak current without any change in the peak potential. The results indicated that a reaction occurred between the positively charged AO and the negatively charged heparin, which led to the decrease of the free AO concentration and subsequent decrease of the reductive peak current of AO. Under the optimal conditions, the decrease of reductive peak current of AO was proportional to heparin concentration in the range of 0.8-6.0 mg L-1 and the data reductive peak current were correlated as Delta ip ''(nA)=807.58 C (mg L-1)-542.51 (n=10, correlation coefficient gamma=0.995) and the detection limit was calculated as 0.13 mg L-1 (3 S-0/S). The new method was successfully applied to the determination of heparin content in heparin sodium injection samples with satisfactory results and good recovery. The interaction of the reactants was also confirmed by UV-Vis absorption spectrophotometric experiments.