DNA is considered to be one of the most promising targets for anticancer agents. Acridine analogues have anticancer activity based on DNA binding and topoisomerases inhibition. However, due to the side effects, resistance and low bioavailability, a few have entered into clinical usage and the mechanisms of action are not fully understood. Novel acridine derivatives are needed for effective cancer therapy. A series of novel 3-nitroacridine-based derivatives were synthesized, their DNA binding and anticancer activities were evaluated. The chemical modifications at position 9 of the 3-nitroacridine were crucial for DNA affinity, thus optimizing anticancer activity. UV–Vis and circular dichroism (CD) spectroscopy indicated interaction of compounds with DNA, and the binding modes were intercalation and groove binding. MTT assay and clonogenic assay showed that compounds 1, 2 and 3 had obvious cell growth inhibition effect. They induced cell apoptosis in human breast cancer cells in a dose-dependent manner, and exhibited anticancer effect via DNA damage as well as cell cycle arrest at G0/G1 phage. Using confocal fluorescent microscope, the apoptotic features were observed. The results suggested that compounds 1–3 with high DNA binding affinity and good inhibitory effect of cancer cell proliferation can be developed as prime candidates for further chemical optimization.