Enzymatic biosensor has been paid much attention to the research fields due to its advantage in medical application. As one of the application, we determined the optimum value of cholesterol oxidase against cholesterol. In this work, we studied the behavior of cholesterol oxidation by enzymatic reaction to get the optimum condition for cholesterol oxidation. The enzyme that used were in two form, free cholesterol oxidase, and immobilized cholesterol oxidase. Cholesterol oxidase was produced from Streptomyces sp. by using solid state fermentation method and identified had high enzyme activity to be 5.12 U/mL. Cholesterol oxidase was simultaneously crosslinked immobilized onto magnetite coated by silicon dioxide (M-SiO2). The support was characterized by Fourier transform infrared (FTIR) to determine the functional group of modified particle and scanning electron microscope (SEM) to observe the morphological or our prepared particle. Cholesterol oxidase sensitivity to substrate was analyzed by using HPLC with different interval time measurements. The oxidation of cholesterol by free enzyme and immobilized enzyme was also investigated. The best sensitivity of cholesterol oxidase was estimated to oxidize Cso (concentration of substrate) 1.46 mM of substrate with Ce (concentration of enzyme) 20 mg/mL for 180 min. Final oxidation value of cholesterol by immobilized enzyme was greater than 60%. The results of this study revealed that immobilized enzyme for cholesterol oxidation was stable, reproducible, and sensitive.