Abstract: In order to study the relationship between the structure and cytotoxicity of the amphoteric surfactants ,MTT assay is adopted to study of proliferation inhibiting effect of betaine amphoteric surfactants, including: four alkyl dimethyl propyl sulphobetaines and three alkyl dimethyl hydroxypropyl sulphobetaines on HeLa cells , CHO cells and CCC-ESF-1 cells . The results indicated that the proliferation inhibiting effect of the chemicals on the cells above gradually become larger with their concentration increased；Proliferation inhibiting effect of all the compounds increased with the growing in number of carbons. Cytotoxicity of alkyl dimethyl propyl sulphobetaine is lower than alkyl dimethyl hydroxypropyl sulphobetaine.