In this work, a new kind of N, Cu codoped carbon dots (N/Cu-CDs) was prepared via a facile one-pot hydrothermal method by using citric acid monohydrate, copper acetate monohydrate and diethylenetriamine. The prepared N/Cu-CDs with a high quantum yield (50.1%) showed excitation-independent emission at 460 nm. The structure and fluorescence properties of N/Cu-CDs were characterized by high-resolution transmission electron microscopy, fluorescence spectrofluorometer, FT-IR spectrometer, UV-visible spectrophotometer and X-ray photoelectron spectroscopy. N/Cu-CDs were applied to establishing a ratiometric fluorescence probe toward HO based on the inner filter effect (IFE) between N/Cu-CDs and DAP (2,3-diaminophenazine, the oxidative product of o-phenylenediamine (OPD)), and provided a ratiometric fluorescence universal platform for detection of the metabolites participating in HO-generation reactions (cholesterol and xanthine). The proposed method was demonstrated to be ultrasensitive and highly selective for cholesterol and xanthine assay with detection limits of 0.03 and 0.10 μM, respectively. The fluorescence probe built was applied to the determination of cholesterol and xanthine in human serum with satisfactory results.