Supported lipid bilayers (SLBs) are simplified model membrane systems that mimic the fundamental properties of biological cell membranes and allow the surface-sensitive tools to be used in numerous sensing applications. SLBs can be prepared by various methods including vesicle fusion, solvent-assisted lipid bilayer (SALB), and bicelle adsorption and are generally composed of phospholipids. Incorporating other biologically relevant molecules, such as cholesterol (Choi), into SLBs has been reported with the vesicle fusion and SALB methods, whereas it remains unexplored with the bicelle absorption method. Herein, using the quartz crystal microbalance-dissipation (QCM-D) and fluorescence microscopy techniques, we explored the possibility of forming SLBs from Chol-containing bicelles and discovered that Chol-enriched SLBs can be fabricated with bicelles. We also compared the Chol-enriched SLB formation of the bicelle method to that of vesicle fusion and SALB and discussed how the differences in lipid assembly properties can cause the differences in the adsorption kinetics and final results of SLB formation. Collectively, our findings demonstrate that the vesicle fusion method is least favorable for forming Chol-enriched SLBs, whereas the SALB and bicelle methods are more favorable, highlighting the need to consider the application requirements when choosing a suitable method for the formation of Chol-enriched SLBs.