The invention provides a preparation method of polyamino acid, which comprises the following steps: dissolving a terminal-aminated hydrophilic polymer, L-cystine-N-endo-carboxylic acid anhydride and amino acid-N-endo-carboxylic acid anhydride in an organic solvent, stirring and reacting, thus obtaining the polyamino acid. The invention further provides polyamino acid nano-hydrogel which comprises the polyamino acid prepared by the method adopting the technical scheme, and water. The polyamino acid which can form the nano-hydrogel can be prepared by only one step, and the step is simple, convenient and fast. The obtained polyamino acid comprises a hydrophilic polymer and a cross-linked polyamino acid, the nano-hydrogel can be formed by dissolving the polyamino acid in the water, and the nano-hydrogel can be used as carrier materials for medicament transmission, and medicament control and release. The terminal-aminated hydrophilic polymer, the L-cystine-N-endo-carboxylic acid anhydrideand the amino acid-N-endo-carboxylic acid anhydride are used as raw materials, and the nano-hydrogel formed by the obtained polyamino acid has good biocompatibility and biodegradability.