Autofluorescence　background　in　complex　biological　samples　is　a　major　challenge　in　achieving　high　sensitivity　of　fluorescence　immunoassays　(FIA).　Here　we　report　an　X-ray　luminescence-based　immunoassay　for　high-sensitivity　detection　of　biomarkers　using　X-ray　scintillating　nanotags.　Due　to　the　weak　scattering　and　absorption　of　most　biological　chromophores　by　X-ray　excitation,　a　low-dose　X-ray　source　can　be　used　to　produce　intense　scintillating　luminescence　from　the　nanotags　for　autofluorescence-free　biosensing.　To　demonstrate　this　concept,　we　designed　and　synthesized　NaGdF4:Tb@NaYF4　core/shell　nanoparticles　as　kind　of　high-efficiency　X-ray　scintillating　nanotags,　which　are　able　to　convert　high-energy　X-ray　photons　to　visible　light　without　autofluorescence　in　biological　samples.　Notably,　strong　X-ray　absorption　and　minimized　surface　quenching　arising　from　the　heavy　Gd3+/Tb3+　atoms　and　core/shell　architecture　of　the　nanoparticles　were　found　to　be　critically　important　for　high-efficiency　X-ray　excited　luminescence　for　high-sensitivity　biosensing.　Our　method　allows　for　sensing　alpha-fetoprotein　(AFP)　biomarkers　with　a　detection　limit　down　to　0.25　ng/mL.　Moreover,　the　as-described　X-ray　luminescence　immunoassay　exhibited　an　excellent　biological　specificity,　high　stability,　and　sample　recovery,　implying　an　opportunity　for　applications　in　complex　biological　samples.　Consequently,　our　method　can　be　readily　extended　for　multiplexing　sensing　and　medical　diagnosis.　©　2018　American　Chemical　Society.