In our pursuit to expand new potential anticancer leads, a series of eighteen novel 1-cyclopropyl6-fluoro-4-oxo-7-(4-substituted piperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid analogues have been synthesized, characterized and evaluated anti-proliferative activity against five human cancer cell lines such as A549 (lung cancer), Mia Paca (pancreatic cancer), HeLa (cervical cancer), MDA MB-231 (breast cancer), MCF7 (breast cancer) and normal embryonic kidney cell line (HEK) were carried out using MTT assay. Few of the synthesized analogues exhibited potent anticancer activity against the cancer cell lines at a lower concentration. The synthesized compounds showed the less toxic effect on normal human embryonic kidney cell line (HEK)compared with doxorubicin. Noticeably, compound 3o exhibited potent activity against all five cancer cell lines compared with ciprofloxacin. Further study exposed that compound 3o could competently intercalate into calf thymus DNA to form 3o-DNA complex which might block DNA replication to apply anti-proliferativeactivity. Docking simulation studies supported by molecular interactions with DNA type II topoisomerase. These derivates can become lead structures for the development of potential anticancer drugs.