In this study, the fluorescence analysis was used to reveal the interaction between berberine derivatives and plasmid DNA. The results showed that berberine (C0) and its 8-alkyl derivatives can enhance the fluorescent intensity of plasmid DNA. Compared with 8-dodecyl- (C12) and 8-hexadecyl- (C16) berberine, 8-alkylberberine with shorter alkyl group, such as 8-ethyl (C2), 8-butyl (C4), 8-hexyl (C6), and 8-octyl (C8) berberine derivatives showed higher fluorescence increasing effect. Among all compounds, C4 showed highest fluorescence increasing effect. All compounds tested obviously enhanced fluorescent intensity at the concentration of 6.25 × 10−5 mol/L. These results suggested that berberine and its derivatives can be selectively inserted to the grooves running down the plasmid DNA helix, thus, lead to the increase of fluorescence intensity of the reaction system. Also, adding proper length of aliphatic chain to berberine could promote the interaction between DNA and berberine derivatives. The results of this study may lay some useful foundation for the development of berberine-based medicine agents.