Iron proteins are of great scientific interest due to their importance as an excellent biomarker for human diseases. Ferritin (Fe3+), being an iron-rich blood protein, is related to various diseases like anemia and cancer. For the first time, we have developed a highly sensitive and selective ferritin biosensor based on fluorescent oxygen-deficient zinc oxide nanosheets through hydrothermal and probe-ultrasonication combined methods. The fluorescence study showed an intense bluish-green fluorescence at λex = 370 nm, after optimization at different excitation wavelengths. In addition, the fluorescence of ZnO1-x nanosheets can be efficiently quenched due to electron transfer reactions in order to achieve quantification analysis. The limit of detection (LOD) was calculated to be 0.015 nM (7.2 ng mL-1) with high linearity (R2 = 0.9930). In addition, the real-world application of the proposed biosensor has been performed on human blood serum samples in the presence of various interfering analytes showing high selectivity and sensitivity with a regression value R2 = 0.9980 indicating the current approach is an excellent biosensor platform.