The fluorescence polarization of a fluorescence probe embedded in vesicle bilayers was assessed for dicarboxylic acid surfactant/dimyristoyl phosphatidylcholine (DMPC) mixtures. The effects of additive surfactants on membrane fluidity were investigated. Simultaneously, mixed vesicles size was estimated from light scattering and vesicle formation was confirmed by electron microscopic observation. With increase in hydrocarbon chain length of additive surfactants, transition temperature of membrane fluidity increased. The transition temperature rose in an order, N-acyl-L-aspartic acid (C_nAsp) /DMPC, alkenylsuccinic acid (C_nSA) /DMPC, and fatty acid/DMPC systems. For the C₁₄SA/DMPC mixed system, size was not affected by mole fraction of a surfactant, while transition temperature varied with the addition of C₁₄SA. For the C₁₂Asp/DMPC system, transition temperature showed no change but particle size remarkably decreased with mole fraction.