The work aims to optimize the optimum culture conditions and methods for production of bacterial pellicle with acetic acid bacteria to obtain a natural and safe production process of pellicle for food packaging. Astringent persimmon vinegar was used as raw material, and excellent acetic acid bacteria with high yield membrane were isolated and screened. The optimum culture method and technological conditions of acetic acid pellicle were optimized by single factor and Plackett−Burman experimental design. The pellicle producing acetic acid bacteria was isolated and purified from astringent persimmon vinegar and identified as gluconacetobacter xylinum. The optimum conditions for culture of pellicle with acetic acid bacteria were as follows:sucrose 5% (mass fraction), peptone 0.7% (mass fraction), yeast extract 1.2% (mass fraction), sodium citrate 0.15% (mass fraction), K2HPO4 0.16% (mass fraction), acetic acid 1% (v/v), absolute ethanol 2% (v/v), seed age 32 h, inoculation amount 9%, cultured for 5 days. The average yield of pellicle was 364.65 g/L, which was 1.48 times as high as that before optimization (246.03 g/L). The average moisture content of wet pellicle was 99.38%, and the rehydration rate was 71.09%-83.39%. The rehydration rate of dry membrane was high and the rehydration speed was fast. The water content can reach more than 70% after one minute of rehydration. With the optimized culture medium and method, high-quality pellicle can be produced with acetic acid bacteria.