Four new naphthyridine derivatives (R1-R4) possessing amino acid or boronic acid moieties have been synthesized and characterized using 1 H and 13 C NMR, FT-IR, and mass spectral techniques. The mechanism of binding of these probes with calf thymus DNA (CT-DNA) has been delineated through UV-Vis, fluorescence, and circular dichroism (CD) spectral techniques along with thermodynamic and molecular docking studies. Small hypochromicity in absorption maximum of the probes without any shift in wavelength of absorption suggests groove binding mode of interaction of these probes with CT-DNA, confirmed by CD and 1H NMR spectral data competitive binding assay with ethidium bromide (EB). CT-DNA quenches the fluorescence of these probes via a static quenching mechanism. In the case of R1 and R4, the observed ΔH o < 0 and ΔS o > 0suggest that these probes interact with CT-DNA through H-bonding and hydrophobic interactions, while in the interaction of R2 and R3, van der Walls and H-boding forces are found to be dominant (ΔH o < 0 and ΔS o < 0). Results of molecular docking investigations corroborate well with that of spectral studies, and these probes bind in the minor groove of DNA. These probes are found to be effective fluorescent staining agents for DNA in agarose gel in gel electrophoresis experiment with sensitivity comparable to that of EB, and DNA amounts as low as 37.5 ng are visually detectable in the gel.