A　series　of　double-chained　zwitterionic　sulfobetain　amphiphiles　(denoted　as　2C16　NnSO3,　n　=　2,　3,　4,　and　6)　having　different　interionic　distances　(spacer)　was　synthesized　and　investigated　by　means　of　surface　pressure　(π)　-　average　molecular　area　(A)　isotherms.　The　influences　of　the　spacer　on　the　2C16　NnSO3　monolayer,　the　2C16　NnSO3/dipalmitoylphosphatidylcholine　(DPPC)　binding　and　the　2C16　NnSO3/DNA　complex　were　studied.　The　monolayers　showed　obvious　different　interfacial　behavior　on　pure　water　subphase,　while　the　properties　of　the　DPPC/2C16　NnSO3　mixed　monolayers　are　independent　of　the　spacer　length.　Few　interactions　between　2C16　NnSO3　and　DNA　were　observed　when　n　<　6　until　divalent　magnesium　ions　(Mg2+)　was　added.　In　comparison,　significant　effect　of　DNA　on　the　isotherms　of　2C16N6SO3　is　observed.　The　results　demonstrated　that　both　the　spacer　length　played　an　important　role　in　the　interfacial　behavior　of　zwitterionic　amphiphiles.　©　Taylor　&　Francis　Group,　LLC.