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Drug-loading transfersomes were prepared with itraconazole, a lipophilic drug, as a model drug to investigate the key factor affecting transfersomes quality and to evaluate their qualities. Drug-loading transfersomes were prepared using film dispersion method. The quality of transfersomes was evaluated by HPLC, transmission electron microscope, particle size analyzer and in vitro release. Itraconazole transfersomes was transparent solution in ivory white color with a mean entrapment efficiency of about 80%. The shape of hollow vesicles was spheroidal with the diameter of approximately 100 nm, and the zeta potential of 45 mV, which had a good transdermal effect. It can be concluded via single-factor investigation that the quality of transfersomes is significantly affected by solvent, salt ion concentration and homogenization pressure and so on. The preparation method obtained through screening and optimizing formulation and technology is feasible and the quality can be controlled.