Bioflocculation is a promising method of removing GO from the water as it is safe, biodegradable, and non-secondary pollution, but few studies on effective and economic bioflocculant of GO have been reported. Accordingly, this work evaluated flocculation efficiency of an extracellular bioflocculant named MBF-21 produced by Enterococcus faecalis. Optimum culture conditions for bioflocculant production were initial medium pH of 7 and incubation temperature of 40ºC. The optimum carbon and nitrogen sources for bioflocculant production were glucose and beef extract. Results of Fourier transform infrared (FTIR) indicated the presence of carboxyl, hydroxyl, and amide groups on the MBF-21. The Zeta potential of MBF-21 analysis revealed that MBF-21 was mainly negatively charged. MBF-21 showed a significant ability in flocculation of GO, and flocculation efficiency was over 90% under neutral, acidic, and alkaline conditions, indicating that flocculation of GO was ion-independent. The conditions for flocculation of GO were optimized by response surface methodology (RSM) and determined to be an 11.57 mg/L dose and flocculation time of 75 min.