Abstract A stable truncated 2’ fluoro-RNA aptamer contributes to the upstream interruption of the blood coagulation pathway by selectively binding with factor IX protein (FIX). This work followed a rational method for virtually analyzing structural orientation and binding interactions between aptamer and FIX. Three possible aptamer models from the same sequence were designed and evaluated for optimum binding with FIX. Molecular docking was performed to find a prime docking domain and the best docking conformation for a stable aptamer-FIX complex. Possible interactions are observed at PRO126-U9, LYS125-U9, LYS122-A7, ARG116-U9, GLY102-G22, THR101-G22, GLN74-G6, GLY60-G5, CGU36-U11, CGU40-A12, and ASP49-A10. A molecular dynamic simulation assessed the strength of the interaction between aptamer and FIX. It was revealed that the aptamer was effectively bound with the average amino acid fluctuation of 1 A. The complex reached equilibrium within ten ns during the initial phase of the simulation and then remained stable over 100 ns. B factor analysis showed a moderate atomic displacement from the crystal structure, except at N-terminal from atom 2247-5938. Principal component analysis of post-dynamic trajectories further revealed the stability by the arrayed orientation of amino acids dihedral angles. Overall, 2’ fluoro-RNA aptamer exhibits stronger stability in their interaction with FIX. Noteworthy, the aptamer's stability as an inhibitor to deactivate the clotting cascade by specifically binding with epidermal growth factor-like (EGF) 1 and 2 domains was evidenced.