In this study, the use of rehydrated whey powder (RWP) solutions containing different carbon sources (citric acid, ammonium sulfate, or glutamic acid) for γ-poly(glutamic acid) (γ-PGA) by Bacillus subtilis ATCC 6633 was explored. After 72 h of fermentation at 30 o C, cell growth, γ-PGA production, molecular weight by SDS-PAGE, rheological properties and NMR analysis of γ-PGA were determined. The growth of B. subtilis was significantly different during 72 h of fermentation in RWP, medium E, and RWP containing citric acid or glutamic acid. These results showed the dynamic viscosity of the 1% polymer solution was 2.5 Pa•s at 10 1/sec shear rate. The maximum γ-PGA concentration was 1.57 g/100 mL in RWP containing glutamic acid, citric acid and ammonium sulfate. Therefore, RWP as a waste product with being efficient and more economic fermentation medium to produce γ-PGA can be ideal for the industrial production.