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The advancement of functional nanomaterials has promoted the development of biomarker sensors underpinning promising analytical tools for a range of bioanalytes such as cholesterol. In this work, we established a light-on fluorescent probe for cholesterol in human serum by coordination of H2O2 on the surface of praseodymia nanorods (Pr6O11 NRs). The distinctive interactions of various nucleotides and H2O2 with praseodymia were examined, whereby good fluorescent quenching and recovery capability were observed. A highly sensitive and selective cholesterol detection was achieved in serum samples with a detection limit of 0.1 μM and recovery of 97.2–101.3%, respectively, due to the high oxygen mobility of praseodymia. The result suggests strong potential for work towards a key probe for a portable clinical test system for cholesterol as well as other H2O2-deriving biomarkers, potentially addressing the ever-increasing demand for the prevention of cardiovascular disease.