Moxifloxacin (MOX) is a fourth-generation synthetic fluoroquinolone antibacterial agent with many important therapeutic properties. Fluorescence quenching was used to study the interaction of MOX with calf thymus DNA (ct-DNA) in aqueous solution. The intercalative binding mode and a static quenching mechanism were confirmed by the Stern-Volmer quenching rate constant (K-q) of 3.48 x 10(11) M-1 s(-1) at 298 K. The thermodynamic parameters (Delta H = -118.4 KJ mol(-1) and Delta S = -299.4 J mol(-1) K-1) were calculated at different temperatures, and they indicate that the main forces between MOX and ct-DNA are hydrogen bonding and Van der Waals force. We proved at the same time the presence of one single binding site on ct-DNA, and the binding constant is 1.28 x 10(5) M-1 at physiological pH. The results may provide a basis for further studies and clinical application of antibiotics drugs.