Liposomes are considered an ideal biomimetic environment and are potential functional carriers for important molecules such as steroids and sterols. With respect to the regulation of self-assembly via sterol insertion, several pathways such as the sterol biosynthesis pathway are affected by the physicochemical properties of the membranes. However, the behavior of steroid or sterol molecules (except cholesterol (ChI)) in the self-assembled membranes has not been thoroughly investigated. In this study, to analyze the fundamental behavior of steroid molecules in fluid membranes, CM, lanosterol, and ergosterol were used as representative sterols in order to clarify how they regulate the physicochemical properties of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes. Membrane properties such as surface membrane fluidity, hydrophobicity, surface membrane polarity, inner membrane polarity, and inner membrane fluidity were investigated using fluorescent probes, including 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, 8-anilino-1-naphthalenesulfonic acid, 6-propionyl-2-(dimethylamino) naphthalene, 6-dodecanoyl-2-dimethylaminonaphthalene, and 1,6-diphenyl-1,3,5-hexatriene. The results indicated that each sterol derivative could regulate the membrane properties in different ways. Specifically, Chl successfully increased the packing of the DOPC/Chl membrane proportional to its concentration, and lanosterol and ergosterol showed lower efficiencies in ordering the membrane in hydrophobic regions. Given the different binding positions of the probes in the membranes, the differences in membrane properties reflected the relationship between sterol derivatives and their locations in the membrane.