A series of double-chained zwitterionic sulfobetain amphiphiles (denoted as 2C16 NnSO 3 , n ¼ 2, 3, 4, and 6) having different interionic distances (spacer) was synthesized and investigated by means of surface pressure (p) -average molecular area (A) isotherms. The influences of the spacer on the 2C16 NnSO 3 monolayer, the 2C16 NnSO 3 /dipalmitoylphosphatidylcholine (DPPC) binding and the 2C16 NnSO 3 /DNA complex were studied. The monolayers showed obvious different interfacial behavior on pure water subphase, while the properties of the DPPC/2C16 NnSO 3 mixed monolayers are independent of the spacer length. Few interactions between 2C16 NnSO 3 and DNA were observed when n < 6 until divalent magnesium ions (Mg 2þ ) was added. In comparison, significant effect of DNA on the isotherms of 2C16N6SO 3 is observed. The results demonstrated that both the spacer length played an important role in the interfacial behavior of zwitterionic amphiphiles.