Cholesterol plays an important role in biological systems, and the quantity of cholesterol in the human body serves as a diagnostic marker for a range of disorders. This article presents a localized surface plasmon resonance (LSPR) sensor based on dual-tapered optical fiber (DTOF) that can detect cholesterol levels in the human body. To stimulate the LSPR effect and increase the sensitivity of the sensing probe, gold nanoparticles were fixed on the DTOF’s surface. In this study, the reaction between cholesterol and cholesterol oxidase altered the refractive index near the sensing probe, and the related spectrum was obtained. In addition, the sensor’s performance, including linear range, repeatability, reusability, stability, and selectivity, was examined. The experimental results indicate that the proposed DTOF-based LSPR sensor is capable of reliably measuring cholesterol levels and has promising biomedical applications.