Accurate　quantitation　of　trace-level　ATP　biomarkers　often　suffers　from　interference　of　sample　matrix　and　remains　a　challenge.　Herein,　a　novel　magnetic　biological　nanosensor　(MBNS)　integrating　tetrahedral　framework　nucleic　acid　(tFNA)-confined　aptamers　and　ultrasensitive　laser-induced　fluorescence　(LIF)　was　tailored　and　evaluated.　The　fluorescent　aptamer　nanoprobe　hybridized　with　FAM-labelled　cDNA　was　particularly　incorporated　into　the　inner　cavity　of　tFNA,　enabling　a　dual-mode　mechanism　that　integrates　molecular　size　selection　and　aptamer　affinity　recognition.　By　this　strategy,　the　benefits　of　tFNA　molecular　sieve,　the　super　sensitive　properties　of　LIF,　and　the　specific　recognition　of　＇aptamer　gene　codes＇　were　integrated,　as　triggered　the　superior　response　of　ATP.　The　structures,　properties　and　reaction　mechanism　of　tFNA,　as　well　as　tFNA　modified　with　aptamer　probe　at　different　sites,　were　characterized　in　detail.　A　high　level　of　anti-interference　was　achieved,　shielding　against　non-specific　adsorption　even　in　matrices　with　high　protein　and　analogue　content,　103∼105　times　higher　than　ATP.　The　release　of　fluorescent　beacon　from　aptamer　probe　was　facilely　driven　by　specific　binding　of　ATP,　and　an　ultra-sensitive　detection　of　75　pmol/L　ATP　was　obtained　without　amplification.　It　was　2–3　orders　of　magnitude　more　sensitive　than　most　fluorescent　aptasensors.　Applied　to　serum　samples,　the　stability　and　reproducibility　of　the　resultant　aptasensor　have　been　validated　with　the　intra-　and　inter-assay　RSD　in　1.6–6.4　%　and　-1.5–8.4　%,　respectively.　Acceptable　recoveries　of　96.5–106.8　%　were　achieved,　consistent　with　the　LC-MS　method.　It　was　expected　to　open　a　simple　and　efficient　protocol　for　highly　specific　and　ultrasensitive　ATP　detection　in　clinical　diagnostics.　©　2024　Elsevier　B.V.