Biotin,　as　a　key　nutrition　fortifier　in　infant　formula,　is　of　vital　significance　for　guaranteeing　the　healthy　growth　of　infants　and　young　children.　The　accurate　detection　of　biotin　has　therefore　become　a　crucial　priority　for　quality　assurance　in　infant　formula.　However,　the　current　bioassay　techniques　encounter　a　series　of　limitations　such　as　cumbersome　procedures　and　low　sensitivity.　Herein,　we　develop　a　sensitive　time-resolved　fluorescence　resonance　energy　transfer　(TR-FRET)　nanoprobe　by　employing　LiLuF4:　Ce/Tb　nanoparticles　(NPs)　as　energy　donors　for　biotin　detection.　By　taking　advantage　of　the　distinctive　millisecond-range　photoluminescence　of　the　NPs,　the　short-lived　background　autofluorescence　can　be　completely　eliminated,　which　enables　the　sensitive　detection　of　biotin　in　infant　formula　within　a　range　of　0–6104　pM　and　a　detection　limit　down　to　0.84　pM　(0.21　pg　mL−1).　This　value　indicates　2−4　orders　of　magnitude　improvement　over　conventional　bioassay　methods.　The　proposed　TR-FRET　biosensor　is　independent　of　expensive　instruments　and　can　be　fully　compatible　with　commercial　microplate　readers.　Specifically,　the　homogeneous　assay　design　enables　rapid,　user-friendly　analysis,　completing　the　entire　detection　process　within　20　min.　These　findings　offer　new　opportunities　towards　rapid　and　sensitive　detection　of　biotin　in　infant　nutrition　products,　thereby　demonstrating　the　great　potential　of　lanthanide　nanoprobes　in　food　safety　monitoring　applications.　©　2025　Elsevier　B.V.