Nine novel naphthalimide-polyamine conjugates were synthesized and their structures were confirmed by elemental analysis, 1 H NMR, 13 C NMR and MS techniques. Their antitumor activities in vitro using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay were assessed on leukemia cells (K562), human breast cancer cells (MB-231) and human hepatoma cells (7721). The results showed that most of them exhibited antitumor activities. Especially, compound 5a was better than amonafide which has been in phase III clinical trials. Fluorescence data revealed that the fluorescence quenching of herring sperm DNA-EB complex by compound 5a was a static quenching type and naphthalimide-polyamine conjugates intercalate into the DNA base pairs.