A composite of citrate-modified β-cyclodextrin (CIT-BCD) and Fe3O4 was prepared by coprecipitation for a non-enzymatic cholesterol sensor application. Characterization using X-ray diffraction spectroscopy confirmed the spinel structure of Fe3O4, whereas infrared spectroscopy indicated that the composite of CIT-BCD@Fe3O4 was successfully synthesized. The cholesterol was detected on the basis of the competition of the inclusion complex formation between β-cyclodextrin (BCD) and cholesterol, and between BCD and methylene blue (MB), and the magnetic particles of Fe3O4 were used as the support medium of BCD. BCD was modified with citrate to improve its cholesterol loading capacity for a computational study through molecular docking simulation, which confirmed that cholesterol formed higher complex stabilities with both BCD and CIT-BCD than with MB, with the ΔGbinding values, of the complexes being −6.4 and −5.7 kcal/mol, respectively. A ratio of 3% (w/w) CIT-BCD@Fe3O4 nanocomposite and a contact time of 10 min were then found as optimum conditions. Furthermore, amperometric measurements performed using a screen-printed carbon electrode at an applied potential of −0.43 V (vs Ag/AgCl) with a measurement time of 90 s was conducted to detect the MB released from the system. Amperometry results showed good linearity (R2 > 0.99) in the cholesterol concentration range of 0–100 μM with an estimated limit of detection of 3.93 μM. Good selectivity towards ascorbic acid, palmitic acid, tyrosine, and threonine was observed, whereas a significant change in current response was found in the presence of arginine. The developed method was successfully demonstrated to determine cholesterol levels in commercial corned beef samples. The method was also successfully validated by HPLC, indicating that the developed sensor is promising for real applications in cholesterol detection, especially in the matrix of meat samples.