Fludarabine is a purine derivative, anti-neoplastic drug and is still being used in the treatments of chronic lymphocytic leukemia, small lymphocytic lymphoma, acute myeloid leukemia, Non-Hodgkin’s lymphoma. It achieves its function by interacting with DNA. Therefore, the binding interactions of such drugs with deoxyribonucleic acid (DNA) is an important subject for pharmaceutical and biochemical studies aiming at designing better DNA binding drugs. Although DNA binding mode of some of the anti-neoplastic drugs has been studied, DNA interaction of Fludarabine has not been explored yet. For this reason, this work has been dedicated to deciphering the experimental and theoretical investigation of Fludarabine binding mechanism via multispectroscopic techniques including UV absorption spectroscopy, thermal denaturation, fluorescence and FTIR spectroscopy, electrochemical and viscosity measurement methods as well as with molecular docking studies under physiological conditions. We observed in the lowest energy docking poses that Fludarabine binds to DNA via major groove binding mode. The nonplanar and extended structure and hydrogen bonding interactions of Fludarabine with the Adenine-Thymine base-pair played a very decisive role in the binding mode as supported by the experimental results.