Optical　biosensors　that　enable　highly　sensitive　detection　of　biomolecules　are　useful　for　applications　in　early　disease　diagnosis.　However,　the　presence　of　UV-vis-induced　background　fluorescence　in　biological　samples　is　still　challenging.　Thanks　to　the　weak　scattering　and　nearly　no　absorption　of　biological　chromophores　under　X-ray　excitation,　we　describe　the　development　of　an　X-ray　nanocrystal　scintillator-based　aptasensor　that　is　able　to　achieve　sensitive　and　homogeneous　detection　of　target　biomolecules.　In　this　work,　aptamer-labeled　lanthanide-doped　nanocrystal　scintillators　was　designed　to　rapidly　and　sensitively　detect　lysozyme　via　fluorescence　resonance　energy　transfer　(FRET)　in　human　serum　samples.　Benefiting　from　the　use　of　low-dose　X-ray　as　an　excitation　source　and　high-efficiency　luminescence　of　heavy　atoms-contained　nanocrystals,　the　proposed　X-ray　nanocrystal　scintillator-based　aptasensor　can　readily　detect　lysozyme　with　a　high　sensitivity　up　to　0.94　nM,　as　well　as　an　excellent　specificity　and　sample　recoveries.　Thus,　our　technique　suggests　that　the　X-ray　scintillating　aptasensor　can　create　a　new　generation　of　autofluorescence-free　high-sensitivity　strategy　for　biomarker　sensing　in　biomedical　applications.　©　2019　American　Chemical　Society.