The invention relates to a preparation method of water stability gamma-polyglutamic acid nanometer fibers. The preparation method comprises the steps of: (1) preparing trifluoroacetic acid water solution of which the mass percentage concentration is 5%-10%, dissolving gamma-polyglutamic acid in the trifluoroacetic acid solution and stirring the solution for 6-8 hours to obtain gamma-polyglutamic acid solution; (2) spinning the gamma-polyglutamic acid solution via static spinning to obtain gamma-polyglutamic acid nanometer fibers; and (3) dissolving the gamma-polyglutamic acid nanometer fibersand EDC (ethylene dichloride) in methanol to have a reaction at room temperature for 3-5 hours, adding cystamine dihydrochlorid to have a reaction for 48-72 hours after the reaction is ended, rinsing, freezing and drying the products after the reaction of cystamine dihydrochlorid is ended, thus obtaining gamma-polyglutamic acid nanometer fibers with good water stability. The preparation method ofthe water stability gamma-polyglutamic acid nanometer fibers is simple and convenient in operation and has moderate technique conditions; the raw materials are easy to obtain, and the prepared gamma-polyglutamic acid nanometer fibers have good biocompatibility so the gamma-polyglutamic acid nanometer fibers have latent application merits in the field of the histological engineering bracket.