The influence of copper(II) ions on the binding of Pazufloxacin (PFLX), which is a forth generation quinolone antimicrobial agent, to calf thymus DNA (CTDNA) has been investigated by fluorescence, absorption spectroscopy and viscosity measurement. The results show that PFLX and Cu(2+) can form binary complex with mole ratio 2:1, PFLX and CTDNA can form complex mediated by Cu(2+). A ternary system of PFLX-Cu(2+)-DNA were proposed. The experimental results show that the PFLX binds to CTDNA by two kinds of binding modes, intercalative mode and groove binding mode.