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.