The multifunctional Schiff base ligand N,N′-bis(5-nitro-salicylaldehyde)azine (NO₂-H₂SALNN) has been successfully synthesized and characterized by ESI-MS⁺, ¹H-NMR spectroscopy and CHN elemental analysis. The very feeble intrinsic fluorescence exerted by the ligand was found to be enhanced by several folds in the presence of methanol (∼168 fold) and water (∼10 fold) molecules. This enhancement of fluorescence intensity is supposed to occur as a result of blocking of rotational isomerization along the azomethine group (CN) due to its coordination with solvent molecules, thereby causing turn on fluorescence at two distinct wavelengths in the presence of methanol and water having a large difference between the two emission wavelengths (Δλ = 125 nm). The hydrogen bond-assisted enhancement fluorescence (HAEF) for methanol and water at two different wavelengths giving two different emissions may be due to the different size/nature of the solvents as well as differences in solvent polarity. The binding of NO₂-H₂SALNN and the solvent molecules was investigated and described by spectroscopic and computational studies. The bioactivity of NO₂-H₂SALNN has also been inspected by DNA binding measurements through spectrometric and thermodynamic studies. DNA binding studies reveal that the ligand interacts with double stranded CT-DNA through a groove binding mode and the intrinsic binding constant was determined by calorimetric studies to be (2.24 ± 0.04) × 10⁵ M⁻¹.