The　advancement　in　miniaturized　Raman　spectrometers,　coupled　with　the　single-molecule-level　sensitivity　and　unique　fingerprint　identification　capability　of　surface-enhanced　Raman　scattering　(SERS),　offers　great　potential　for　point-of-care　testing　(POCT).　Despite　this,　accurately　quantifying　analyte　molecules,　particularly　in　complex　samples　with　limited　sample　volumes,　remains　difficult.　Herein,　we　present　a　versatile　and　reusable　SERS　microplatform　for　highly　sensitive　and　reliable　quantitative　detection　of　adenosine　triphosphate　(ATP)　in　biological　fluids.　The　platform　utilizes　gold-Prussian　blue　core-shell　nanoparticles　modified　with　polyethyleneimine　(Au@PB@PEI　NPs),　embedded　within　gold　nanoparticle-immobilized　capillary-based　silica　monolithic　materials.　PB　acts　as　an　internal　standard,　while　PEI　enhances　molecular　capture.　The　periodic,　bimodal　porous　structure　of　the　silica　monolithic　materials　provides　uniform　and　abundant　sites　for　nanoparticle　attachment,　facilitating　rapid　liquid　permeation,　intense　SERS　enhancement,　and　efficient　enrichment.　The　platform　regulates　ATP　capture　and　release　through　magnesium　ions　in　the　liquid　phase,　eliminating　matrix　interferences　and　enabling　platform　reuse.　Integrating　efficient　molecular　enrichment,　separation,　an　interference-free　internal　standard,　a　liquid　flow　channel,　and　a　detection　chamber,　our　platform　offers　simplicity　in　operation,　exceptional　sensitivity　and　accuracy,　and　rapid　analysis　(∼10　min).　Employing　PB　as　an　internal　calibration　standard,　ratiometric　Raman　signals　(I732/I2123)　facilitate　precise　ATP　quantification,　achieving　a　remarkable　limit　of　detection　down　to　0.62　pM.　Furthermore,　this　platform　has　been　proven　to　be　highly　reproducible　and　validated　for　ATP　quantification　in　both　mouse　cerebrospinal　fluid　and　human　serum,　underscoring　its　immense　potential　for　POCT　applications.　©　2024