The binding properties on [PrL 2 (NO 3 )](NO 3 ) 2 (L=9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperaziny)-7-oxo-7Hpyrido[1,2,3-de]-1,4-benzoxazine-6-carbaldehyde benzoyl hydrazone) to bovine serum albumin (BSA) have been studied for the first time using fluorescence spectroscopy in combination with UV–Vis absorbance spectroscopy. The results showed that [PrL 2 (NO 3 )](NO 3 ) 2 strongly quenched the intrinsic fluorescence of BSA through a static quenching procedure, and non-radiation energy transfer happened within molecules. The number of binding site was about 1, and the efficiency of Förster energy transfer provided a distance of 4.26nm between tryptophan and [PrL 2 (NO 3 )](NO 3 ) 2 binding site. At 288, 298, 310K, the quenching constants of BSA–[PrL 2 (NO 3 )](NO 3 ) 2 system were 5.11×10 4 , 4.33×10 4 and 3.71×10 4 lM −1 . ΔH, ΔS and ΔG were obtained based on the quenching constants and thermodynamic theory (ΔH<0, ΔS>0 and ΔG<0). These results indicated that hydrophobic and electrostatic interactions are the mainly binding forces in the [PrL 2 (NO 3 )](NO 3 ) 2 –BSA system. In addition, the CD spectra have proved that BSA secondary structure changed in the presence of [PrL 2 (NO 3 )](NO 3 ) 2 in aqueous solution. Moreover, the interaction between [PrL 2 (NO 3 )](NO 3 ) 2 and calf thymus DNA (CT DNA) was studied by spectroscopy and viscosity measurements, which showed that the binding mode of the [PrL 2 (NO 3 )](NO 3 ) 2 with DNA is intercalation. The DNA cleavage results show that in the absence of any reducing agent, the [PrL 2 (NO 3 )](NO 3 ) 2 can cleave plasmid pBR322 DNA and its hydrolytic mechanism was demonstrated with hydroxyl radical scavengers and singlet oxygen quenchers.