Bicelles are submicrometer-sized disc-shaped molecular self-assemblies that can be obtained in aqueous solution by dispersing mixtures of certain amphiphiles. Although phospholipid bicelle and phospholipid vesicle assemblies adopt similar lipid bilayer structures, the differences in bilayer characteristics, especially physicochemical properties such as bilayer fluidity, are not clearly understood. Herein, we report the lipid ordering properties of bicelle bilayer membranes based on induced circular dichroism (ICD) and fluorescence polarization analyses using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. Bicelles were prepared by using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), while pure DMPC vesicles and pure DHPC micelles were used as references. At temperatures below the phase transition temperature of DMPC, the bicelles showed lower membrane fluidities, whereas DHPC micelles showed higher membrane fluidity, suggesting no significant differences in bilayer fluidity between the bicelle and vesicle assemblies. The ICD signals of DPH were induced only when the membrane was in ordered (solid-ordered or ripple-gel) phases. In the bicelle systems, the ICD of DPH was more significant than that of the DMPC vesicle. The induced chirality of DPH was dependent on the chirality of the bilayer lipid. Compared to that of the DMPC/DHPC bicelle, the ICD of the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/DHPC bicelle was higher, while that of the bovine sphingomyelin/DHPC bicelle was lower. Because the lipids are tightly packed in the ordered phase, the ICD intensity reflects the molecular ordering state of the lipids in the bicelle bilayer.