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Background: Among diverse protein purification systems, affinity chromatography is the most attractive one in the purification process of coagulation factors. Coagulation factor VII is a plasma serine protease that has a significant role in natural human hemostasis and its recombinant form such as AryoSeven™, has been applied in clinical treatment of bleeding disorders. Immunoaffinity chromatography is the purification method of choice that is currently applied in the development of coagulation factor VIIa products. Aptamers as nucleic acid based affinity ligands are more promising than monoclonal antibodies. In addition, DNA aptamers are more acceptable than RNA ones in this regard. Methods: In this study, two of the aptameric DNA oligonucleotides that showed acceptable affinities for purification of coagulation factor VIIa from plasma, were selected to evaluate their affinity against Aryoseven. A serial dilution of fluorescence labeled aptamers was incubated against the concentration of 1 nM from Aryoseven. Then, a fluorescence index was calculated according to the fluorescence intensity data measured from test and control samples. The dissociation constant of aptamers was calculated according to the fluorescence index using Prism5 software. Results: Results showed that the binding affinity of the 44 nucleotide aptamer was more than 81 nucleotide aptamer sequence. As a result, this aptamer could be optimized in order to develop aptamer based affinity chromatography process for this form of recombinant coagulation factor VIIa. Discussion: Aptamers with shorter length of sequence could show higher affinity in target binding, as they could adapt more easily to suitable conformation according to target interaction. However, it should be considered that the selectivity of affinity ligands is also important for target purification and analytical applications.