Binding　of　a　target　by　a　probe　for　selective　detection　depends　on　the　state　of　the　probes　on　the　sensing　interface.　Here,　the　hanging　strand　length　of　triple-helix　DNA　was　used　to　form　tripod　probes　immobilized　via　pi-pi　interactions　on　a　reduced　graphene-oxide　substrate.　The　spacing　between　the　probes　was　adjusted　by　controlling　the　lengths　of　the　tripod　＂feet＂　on　the　substrate;　that　is,　increased　probe　spacing　occurred　when　foot　size　increased　over　the　range　of　6-12　bases.　The　surface　coverages　and　electron-transfer　rates　mediated　the　tripod　DNA　probes　were　characterized　by　electrochemical　methods　and　atomic　force　microscopy.　The　electron-transfer　mediated　by　the　tripod　DNA　probes　was　higher　than　that　mediated　by　doubled-stranded　DNA.　Then　different　sizes　tripod　DNA　probes　were　developed　for　protein-CEA　detection.　The　DNA　probes　with　10　bases　feet　showed　the　best　detection　limit　of　detection　of　10(-6)　ng/mL　in　the　detection　linear　range　(10(-6)　-25　ng/mL).　The　result　demonstrated　the　tripod　DNA　probes　with　different　sizes　could　obtain　excellent　sensitivity　when　it　applied　to　the　target　with　appropriate　size.　This　interspace-controlled　biosensing　interface　of　tripod　DNA　probes　with　enhanced　charge　transfer　should　find　widespread　applications　in　clinical,　medical,　biological,　and　environmental　areas　for　precise　detection　of　differently　sized　targets.