Rapid　detection　of　heparin-binding　protein　(HBP)　is　essential　for　timely　intervention　in　sepsis　cases.　Current　detection　techniques　are　usually　antibody-based　immunological　methods,　which　have　certain　problems,　such　as　complexity　and　slow　detection,　and　fall　short　in　meeting　the　urgency　of　clinical　needs.　The　application　of　an　aptamer　can　address　these　concerns　well.　In　this　study,　HBP-specific　DNA　aptamers　were　screened　first.　Among　which,　Apt-01,　Apt−02,　and　Apt−13　had　a　high　affinity　for　HBP,　exhibiting　impressive　KD　values　of　3.42,　1.44,　and　1.04　nmol/L,　respectively.　Then,　the　aptamer　of　HBP　and　its　partially　complementary　primer　probe　were　combined　to　form　double-stranded　DNA　(dsDNA)　and　synthesize　a　circular　DNA　template.　The　template　is　complementary　to　the　primer　probe,　but　due　to　the　presence　of　dsDNA,　ExoIII　cleaves　C2-13　as　an　RCA　primer　probe,　rendering　the　template　unable　to　recognize　the　primer　probe　and　preventing　the　RCA　reaction　from　proceeding.　When　the　target　is　present,　it　competes　with　the　adapter　for　recognition　and　releases　C2-13,　exposing　its　3′　end.　After　initiating　the　RCA　at　room　temperature　and　reacting　with　SYBR　GreenII　at　37　°C　for　20　min,　fluorescence　changes　can　be　observed　and　quantitatively　analyzed　at　a　530　nm　wavelength,　achieving　quantitative　biological　analysis.　Apt-01　was　used　to　develop　a　fluorescent　biosensor　for　HBP　detection,　which　exhibited　a　good　linear　range　(0.01　nmol/L　to　10　nmol/L)　and　detection　limit　(0.0056　nmol/L).　This　advancement　holds　the　potential　to　lay　a　solid　groundwork　for　pioneering　sensitive　and　specific　methods　for　HBP　detection　and　to　significantly　enhance　the　diagnostic　processes　for　sepsis.　©　2024　by　the　authors.