Fludarabine is a purine derivative, anti-neoplastic drug and is still being used in the treatments ofchronic lymphocytic leukemia, small lymphocytic lymphoma, acute myeloid leukemia, Non-Hodgkin’slymphoma. It achieves its function by interacting with DNA. Therefore, the binding interactions of suchdrugs with deoxyribonucleic acid (DNA) is an important subject for pharmaceutical and biochemicalstudies 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 thisreason, this work has been dedicated to deciphering the experimental and theoretical investigation of Flu-darabine binding mechanism via multispectroscopic techniques including UV absorption spectroscopy,thermal denaturation, fluorescence and FTIR spectroscopy, electrochemical and viscosity measurementmethods as well as with molecular docking studies under physiological conditions. We observed in thelowest energy docking poses that Fludarabine binds to DNA via major groove binding mode. The nonpla-nar and extended structure and hydrogen bonding interactions of Fludarabine with the Adenine-Thyminebase-pair played a very decisive role in the binding mode as supported by the experimental results.