Cholesterol (CHOL) is an important clinical biochemical indicator that plays an important role in the regulation of the fluidity, permeability, and microstructure of cell membranes. Therefore, it is necessary to accurately monitor CHOL levels in biological samples for the early prevention and diagnosis of various diseases. The single-band red upconversion nanoparticle (UCNP) emits light within the optical transmission window of biological tissues, and can penetrate deeper biological tissues and cause less energy loss due to scattering and thus have higher sensitivity and accuracy. Here, using the nontoxic, sensitive, and photochemically stable 3,3′,5,5′-tetramethylbenzidine (TMB) as the quenching agent and single red UCNP as the fluorescent donor, a dual-readout colorimetric and fluorescent sensor was developed to detect CHOL. The detection mechanism and feasibility were discussed in detail, and experimental conditions such as Fe²⁺ concentration, TMB concentration and reaction time were explored. Under optimal conditions, the limits of CHOL detection by colorimetry and fluorescence were 0.85 μM and 0.63 μM. The sensing system was used to measure CHOL in serum samples and the values obtained by these two modes were close, and the spiked recoveries were 97.2–102.2% and 97.1–103.7%, respectively, which holds great potential in clinical diagnosis and health management.