Cholesterol is the precursor of various steroid hormones, bile acid, and vitamin D with functions related to regulation of membrane permeability and fluidity. However, the presence of excess blood cholesterol may lead to arteriosclerosis and hypertension. Moreover, dietary cholesterol may affect blood cholesterol levels. Generally, cholesterol determination is performed by spectrophotometric or chromatographic methods, but these methods are very time consuming and costly, and require complicated pretreatment. Thus, the development of a rapid and simple analysis method for measuring cholesterol concentration in food is needed. Multi-walled carbon nanotube (MWCNT) was functionalized to MWCNT-$NH_2$ via MWCNT-COOH to have high sensitivity to $H_2O_2$. The fabricated MWCNT-$NH_2$ was attached to a glassy carbon electrode (GCE), after which Prussian blue (PB) was coated onto MWCNT-$NH_2$/GCE. MWCNT-$NH_2$/PB/GCE was used as a working electrode. An Ag/AgCl electrode and Pt wire were used as a reference electrode and counter electrode, respectively. The sensitivity of the modified working electrode was determined based on the amount of current according to the concentration of $H_2O_2$. The response increased with an increase of $H_2O_2$ concentration in the range of 0.5~500 ${mu}M$ ($r^2$=0.96) with a detection limit of 0.1 ${mu}M$. Cholesterol oxidase was immobilized to aminopropyl glass beads, CNBr-activated sepharose, Na-alginate, and toyopearl beads. The immobilized enzyme reactors with aminopropyl glass beads and CNBr-activated sepharose showed linearity in the range of 1~100 ${mu}M$ cholesterol. Na-alginate and toyopearl beads showed linearity in the range of 5~50 and 1~50 ${mu}M$ cholesterol, respectively. The detection limit of all immobilized enzyme reactors was 1 ${mu}M$. These enzyme reactors showed high sensitivity; especially, the enzyme reactors with CNBr-activated sepharose and Na-alginate indicated high coupling efficiency and sensitivity. Therefore, both of the enzyme reactors are more suitable for a cholesterol biosensor system.