In this study, the interactions between three quaternary ammonium salt (QAS) cationic surfactants with different branched-chain lengths (TMBAC, TEBAC, and TBBAC) and DNA are investigated by UV-vis absorption, fluorescence and CD spectroscopy, viscosity method, and gel electrophoresis. Berberine hydrochloride (BR) is utilized as a fluorescent probe. The three interaction modes and strengths are compared. The effects of surfactant concentrations, ratio of DNA and BR, and ionic strength on the interaction are estimated. DNA conformational changes are explored. The results indicate that three surfactants can interact with DNA through electrostatic interaction rather than groove and intercalation binding. The interaction results in DNA double helix compression. Also, interaction strength is TBBAC-DNA >
TEBAC-DNA >
TMBAC-DNA due to different branched-chain lengths. Moreover, fluorescence quenching extent is more obvious at 10.0:1.0 molar ratio (DNA: BR). The fluorescence quenching of three surfactant-DNA-BR systems is static. Three binding models are equal, and three interaction processes are spontaneous. The binding force of TBBAC-DNA is electrostatic, while that of TMBAC-DNA and TEBAC-DNA is Van der Waals forces and hydrogen bonding. Besides, DNA conformation keeps the B-form. It is expected to offer insights into the interaction of QAS cationic surfactants with different branched-chain lengths and DNA.