We report a facile method for the preparation of vesicle-like carbon dots (VCDs) via dry-heating of surfactant solutions. Like most reported CDs, VCDs possess interesting fluorescence properties. Entrapment of enzymes and gold nanoclusters (AuNCs) inside the VCDs allows for the development of fluorescent probes for the quantitation of substrates of interest, with the advantages of high sensitivity and selectivity. The AuNCs act as a probe, and the VCDs as an internal standard confine the AuNCs, enzymes, and analytes to provide high local concentrations to enhance the assay sensitivity. In this study, we employed cholesterol oxidase (ChOX) as a model enzyme for the quantitation of cholesterol. The as-formed H2O2 through the enzyme reaction inside the VCDs causes fluorescence quenching of AuNCs (excitation/emission wavelengths of 320/670 nm), but not that of the VCDs (excitation/emission wavelengths of 320/400 nm). To improve the sensitivity and linearity, the fluorescence ratios of AuNCs/VCDs are plotted against analyte concentration. The present ratiometric fluorescent method allows for the detection of H2O2 over the concentration range of 1–100 μM, with a detection limit of 0.67 μM, and cholesterol concentrations ranging from 5 to 100 μM, with a detection limit of 2.8 μM. The practicality of this fluorescent method has been further validated by evaluating cholesterol levels in human serum samples with sufficient accuracy and recovery, revealing its great prospective in diagnosis and biomedical applications.