Abstract Multilayers of amphiphilic molecules are often prepared by the repeated transfer of a substrate through a Langmuir monolayer at the air/water interface. The Langmuir-Blodgett technique is not a suitable method for the construction of multilayers of cholesterol. In this study, the application of the spin-coating technique to the preparation of cholesterol multilayers was investigated. Scanning force microscopy was used to examine the morphology and thickness of cholestrrol films as a function of the frequency with which the substrate was spun, the concentration of the solution used to spin-coat the substrate, and the type of solvent. It was found that the amount of cholesterol that was deposited on the substrate was proportional to the concentration of the solution and to the inverse of the square root of the spinning frequency. Experiments performed with different solvents showed that, in cholesterol films were deposited on the substrate if the vapour pressure of the solvent was higher than 10 kPa at 298 K. Our results suggest that well-ordered cholesterol multilayers with adjustable thicknesses can be prepared with the spin-coating technique.