The invention relates to a polypeptide-containing asymmetric hyperbranched amphoteric polyelectrolyte which is prepared through a click reaction by utilizing two hyperbranched copolymer segments with same or different algebras and containing different polypeptide chain segments on the tail ends, wherein the hyperbranched copolymer segments have different molecular structures and asymmetries of chemical structures; the hyperbranched copolymer segments are respectively provided with polycation and polyanion as well as the asymmetries of charges; and the polypeptide-containing asymmetric hyperbranched amphoteric polyelectrolyte per se is provided with pH responsiveness to environment. The preparation method comprises the steps of: (1) preparing hyperbranched basal body containing alkyne end group and azido end group; (2) preparing the polypeptide-containing hyperbranched copolymer segments; (3) preparing a polypeptide-containing asymmetric hyperbranched copolymer; and (4) preparing the polypeptide-containing asymmetric hyperbranched amphoteric polyelectrolyte. The invention has the positive effects that: the hyperbranched amphoteric polyelectrolyte provided with asymmetries of chemical structures and charges as well as biocompatibility and biodegradability is prepared and has application value in the fields of biochemical sensors, drug deliveries and gene therapies.