Heparin　is　a　highly　negatively　charged　sulfated　linear　polymer　glycosaminoglycan　that　has　been　widely　used　as　an　anticoagulant　in　medicine.　Protamine　is　a　cationic　protein　rich　in　arginine　that　is　used　to　treat　the　blood-brain　barrier　during　excess　heparin　surgery.　Trypsin　is　the　most　important　digestive　enzyme-encoding　generated　by　the　pancreas　and　can　specifically　cleave　the　carboxyl　ends　of　arginine　and　lysine　residues.　Heparin,　protamine,　and　trypsin　interact　and　constrain　each　other,　and　their　fluctuations　reflect　the　body＇s　dysfunction.　Therefore,　it　is　necessary　to　develop　a　fast,　sensitive,　and　highly　selective　assay　for　regularly　monitoring　the　levels　of　heparin,　protamine,　and　trypsin　in　serum.　Herein,　a　fluorescent　and　colorimetric　dual-mode　upconversion　nanoparticle　(UCNP)　biosensor　was　used　for　the　determination　of　heparin,　protamine,　and　trypsin　based　on　the　oxidase-mimicking　activity　of　Ce4+　and　electrostatic　control.　The　biosensor　exhibited　sensitive　detection　of　heparin,　protamine,　and　trypsin　with　low　limits　of　detection　(LODs)　of　16　ng/mL,　87　ng/mL　and　31　ng/mL,　respectively.　Furthermore,　the　designed　biosensor　could　eliminate　autofluorescence,　which　not　only　effectively　increased　the　accuracy　of　the　sensor　but　also　provided　a　new　sensing　pathway　for　the　detection　of　differently　charged　biotargets.　©　2024　Elsevier　B.V.