This study was designed to determine the optimum conditions of three different factors (mixing time, mixing temperature, and tube size) in reduction of cholesterol in milk using immobilized $eta$-CD beads. Immobilized $eta$-CD glass beads were prepared at different conditions of silaniza-tion and $eta$-CD immobilization reactions. In result, the glass beads (diameter 1 mm) at 20 mM 3-isocyanatopropyltriethoxysilane and 30 mM $eta$-CD without base showed the highest choles-terol removal rate as 41%. Using above immobilized $eta$-CD glass beads, the cholesterol removal rate was 40.2% with 6 h of mixing time in 7 mm diameter tube at $10^{circ}C$. After choles-terol removal from milk, the glass beads were washed for cholesterol dissociation and reused. In recycling study, the cholesterol removal rate was 41%, which was mostly same as that using new glass beads. These results indicated that cholesterol removal rate was about 40% with $eta$-CD immobilized glass beads, however, the recycling efficiency was almost 100%.