NMR studies have indicated that the anti-tumor therapeutic agent actinomycin D (ACTD) can induce seemingly single-stranded DNA (ssDNA) oligomer 5'-CCGTT(3)GTGG-3' to form a hairpin structure with tandem GT mismatches at the stem region next to a loop of three stacked thymine bases. In an effort to uncover the preference of binding sequence and to elucidate the thermodynamics properties of the binding, a combination of spectroscopic techniques and computational simulation studies was performed with d(CCGTT(n)GTGG) and d(CCGAA(n)GAGG) (denoted as GTT(n) and GAA(n), respectively; n = 3, 5, and 7) sequences. In the presence of 7-amino actinomycin D (7AACTD), all the six oligomers formed stable hairpin structures. The GTT(5)-7AACTD/GAA(5)-7AACTD hairpin structure was more stable than the corresponding GTT(5)-7AACTD and GAA(n)-7AACTD (n = 3, 7). No significant AG difference was observed between GTT(n)-7AACTD and GAA(n)-7AACTD complexes with the same loop length. In agreement with the 7AACTD-induced hairpin stability results, the binding affinity of GTT(n) and GAA(n) with 7AACTD increased from n = 3 to n = 5 and then decreased when n is 7. Moreover, GTT(n) and GAA(n) with the same loop length showed comparable binding affinities to 7AACTD. Furthermore, molecular dynamics simulations found that van der Waals interactions between GTT(n)/GAA(n) and 7AACTD were the primary attractive forces for 7AACTD binding, and the electrostatic interactions between the carbonyl groups of 7AACTD and bases in the hairpin were the major unfavorable forces. These findings furthered our understanding that 7AACTD is sensitive to the loop size and sequence as well as tandem GT/GA mismatches of their deoxyribonucleic acid (DNA) targets. A deep understanding of the thermodynamics and the molecular recognition mechanism of 7AACTD with ssDNAs would further the development of ACTD-like antitumor agents.