Poly(γ-glutamic acid) (γ-PGA) is a promising biomaterial with a wide range of unique applications. To extensively screen γ-PGA-producing bacteria with high yield and different molecular weight, we developed an integrated high-throughput strategy. Firstly, γ-PGA-producing bacteria were selected in a primary screen plate containing a basic dye (neutral red) based on the concentric zone formed through the electrostatic interaction between the dye and the secreted acidic polymer γ-PGA. Then, the isolates were cultured in 50 ml tubes instead of 250 ml flasks. A good correlation of fermentation results in 50 ml tubes and 250 ml flasks was observed. Thirdly, the γ-PGA yield and weight-average molecular weight (M (w)) were simultaneously determined by spectrophotomic assay (UV assay) and neutral red plate assay. The results showed that the diameter of the concentric zone varied among isolates and was negatively correlated with the weight-average molecular weight of γ-PGA. The accuracy of the methods was comparable to that of high-performance liquid chromatography and gel permeation chromatography assay. Lastly, γ-PGA obtained from the target isolates was rapidly identified using thin layer chromatography assay. With this strategy, 13 bacteria with high yield and various molecular weights of γ-PGA from 500 obvious single colonies on the primary screen plate were obtained.