The interaction between Rhodamine B and calf thymus DNA（ct-DNA） at physiological conditions were studied by the techniques of absorption spectroscopy, fluorescence spectroscopy and confocal imaging. The results of spectroscopy experiments suggested that the hypochromicity of Rhodamine B could appear in the presence of ct-DNA from 450nm to 650nm in the absorption spectroscopy, and the fluorescence of Rhodamine B was quenched in the presence of ct-DNA from 560nm to 660nm in the fluorescence emission spectroscopy. At the same time，the fluorescence polarization of Rhodamine B varied with the addition of ct-DNA.It was indicated that Rhodamine B could interact with ct-DNA. In the competition experiments, Rhodamine B was unable to replace methylene blue（MB） from MB-ct-DNA complex. It was indicated that the interaction between Rhodamine B and ct-DNA took place by the mode of groove combination. The results of confocal imaging experiments showed that the fluorescence of Rhodamine B was quenched quite obviously after the addition of ct-DNA. HeLa cells were dyed with Rhodamine B and 4′,6-diamidino-2-phenylindole（DAPI）,then used for confocal imaging. It was further verified that the cell nucleus could be dyed red through the groove combination interaction between Rhodamine B and ct-DNA.