Objective To study the interaction between biomacromolecules (protein and DNA) and different Rhizoma Coptidis alkaloids (palmatine, coptisine, epiberberine, jatrorrhizine and berberine) and the difference among 5 Rhizoma Coptidis alkaloids. Methods Bovine serum albumin (0.5 mL of 1.10×10^-5 mol/L) was added to 1.5 mL of each alkaloid of different concentrations (concentrations of palmatine, coptisine, epiberberine, jatrorrhizine and berberine being 0, 1.95×10^-6, 3.9×10^-6, 7.8×10^-6, 1.56×10^-5 and 3.12×10^-5 mol/L, respectively). The reaction system was shaken and incubated at 37℃ for 1 h, and then the emission spectra of mixed solution were recorded within 300 to 500 nm at 280 nm excitation. Plasmid DNA (0.5 mL of 0.1%) was added to 1.5 mL of each alkaloid with different concentrations (concentrations of palmatine, coptisine, epiberberine, jatrorrhizine and berberine being 0, 1.95×10^-6, 3.9×10^-6, 7.8×10^-6 and 1.56×10^-5 mol/L, respectively). The reaction mixture was well shaken, and then the fluorescent emission spectrum was recorded from 480 to 650 nm at 368 nm excitation by an F-4500 fluorescence spectrophotometer. Results All the Rhizoma Coptidis alkaloids in test exhibited similar quenching effect on fluorescence of bovine serum albumin, and they all showed fluorescence enhancing effect on plasmid DNA, and the enhancing effects varied greatly among the five alkaloids, with epiberberine showing the strongest activity, followed by coptisine, palmatine, berberine, and jatrorrhizine in order. The strongest interaction between DNA and five alkaloids were found at the concentration of 7.8×10^-6mol/L. Conclusion Strong interaction can be found between Rhizoma Coptidis alkaloids and biomacromolecules including protein and DNA. The interaction of Rhizoma Coptidis alkaloids with plasmid DNA varies greatly among different alkaloids and is similar with bovine serum albumin. 9,10-methylene-dioxy groups and 2,3-dimethoxy groups might have promoted the interaction between alkaloids and DNA.