We constructed a metabolically engineered glutamate-independent Bacillus amyloliquefaciens strain with considerable γ-PGA production. It was carried out by double-deletion of the cwlO gene and epsA-O cluster, as well as insertion of the vgb gene in the bacteria chromosome. The final generated strain NK-PV elicited the highest production of γ-PGA (5.12 g l−1), which was 63.2% higher than that of the wild-type NK-1 strain (3.14 g l−1). The γ-PGA purity also improved in the NK-PV strain of 80.4% compared with 76.8% for the control. Experiments on bacterial biofilm formation experiment showed that NK-1 and NK-c (ΔcwlO) strains can form biofilm; the epsA-O deletion NK-7 and NK-PV strains could only form an incomplete biofilm.