Ion pair amphiphile, prepared from mixed cationic/anionic surfactants, has a pseudodouble-chain structure and can be used as a raw material to form a vesicular structure, or catanionic vesicle, in aqueous phase. It has been demonstrated that catanionic vesicles could be fabricated from the ion pair amphiphile hexadecyltrimethylammonium-dodecylsulfate (HTMA:DS) by using a forced formation process with the addition of double-chain cationic surfactants, dialkyldimethylammonium bromide (DXDAB), and the physical stability of the HTMA:DS/DXDAB catanionic vesicles could be further improved in the presence of cholesterol. Through the analysis of bilayer fluidity and solvation, as well as the data integration of different analysis methods, combined with molecular dynamics simulations, the complex effects of the added cholesterol on the molecular arrangement and interaction in the bilayer structures of the HTMA:DS/DXDAB catanionic vesicles were elucidated.