Thrombin binding aptamer (TBA) is a 15-mer single-strand DNA that was identified by SELEX screening technology. It adopts a chair-type antiparallel G-quadruplex and can specifically interact with thrombin, thus inhibiting blood coagulation. Isonucleoside (isoNA) is a type of nucleoside isomer in which the base is shifted to 2′-positions of the glycosyl group, endowed with the ability to modulate local conformation of nucleotides, and L-isoNA could alter the conformation more due to the inversion of glycosyl configuration. Incorporation of L-isothymidine (L-isoT) at T3, T9, T12 positions and D-isoT at the T7 position in TBA's loop regions promoted the formation of G-quadruplex, resulting in enhanced affinity with thrombin and an increased anticoagulant effect. Computer simulation indicated that TBA-12L showed the strongest binding with thrombin, which was consistent with experimental results. The bioactivity of double isoNA incorporated TBA with D-IsoT at T7 and L-IsoT at T12 was comparable to that of TBA-12L, suggesting that the T12 of TBA was very important in interaction with thrombin. Our study also suggested that TBA might interact with two thrombin molecules through the TT loops (T3T4, T12T13) and TGT loop, but the second bonding did not show additional biological effects.