This study aimed to develop a simple and low-cost cholesterol detection using UV-vis spectrophotometer with β-cyclodextrin (BCD)/Fe3O4 nanocomposite as the identification agent. Cholesterol detection was achieved by replacing methylene blue (MB) molecules, which formed inclusion complexes in BCD cavities. The results showed that cholesterol concentration could be determined from the released amount of MB detected by UV-vis spectrometer. To enhance the sensitivity of signal responses of the sensors, BCD was functionalized with citrate before being deposited on Fe3O4 magnetic nanoparticles. The characterization of the nanocomposite was performed by X-Ray-diffraction (XRD), Fourier Transform Infrared (FTIR) spectra, and Scanning Electron Microscope-Energy Dispersive X-Ray (SEM-EDX). The developed sensor showed linear responses (R2<0.99) toward cholesterol concentrations in the range between 0 and 100 μM, with a sensitivity of 0.56 x 103 a.u. M-1 and estimated detection limit of 7.71 μM. Comparison with the previous detections using the electrochemical method showed that the performance of the developed sensor was comparable. Furthermore, by applying the optimum parameters, including pH solution of 7.4, contact time of 10 min, and composite weight of 3% containing 3% citrate-modified BCD, the developed sensor successfully analyzed cholesterol concentrations in human serum samples. © (2025), (International Journal of Technology). All rights reserved.