In the present study, a series of new C3-quinazolinone linked ?-carboline conjugates were synthesized successfully and evaluated as DNA intercalative topo I inhibitors. It was found that most of the compounds showed good cytotoxic activity, in particular, compounds 10a, 10e and 10u are exhibited potent cytotoxicity against all the tested four cell lines with IC50 values are ranging from 1.19±0.33 to 5.37±0.28 µM. topo I mediated DNA relaxation assay results showed that these compounds could significantly inhibit the activity of topo I. The structure-activity relationship studies indicate the importance of the substitutions at C1 and C3 positions of the ?-carboline moiety. These compounds induced cell cycle arrest in G2/M phase. Further, spectroscopic studies substantiated the biological activities as well as the nature of interactions with the DNA. The intercalative mode of binding with the DNA was established by several biophysical studies like UV- visible, fluorescence and circular dichroism and viscosity. The results obtained from biophysical studies were further supported by the molecular docking studies.