In the present work, a hyperthermostable superoxide dismutase (SOD) was isolated from Thermus thermophilus HB27 using a membrane-based process after being expressed in E. coli. It was found that a two-stage ultrafiltration process using a 100 kDa MWCO regenerated cellulose (RC) membrane and a 50 kDa MWCO polyethersulfone (PES) membrane could successfully employed to isolate SOD from the crude feedstock. The effects of solution pH, ionic strength and permeate flux on the transmission of proteins were quantified using parameter scanning ultrafiltration. Under optimized conditions, the purity of SOD obtained was 96% with a sepcific activity of 1412 U/mg protein and the recovery of SOD from the feedback was close to 87%. The enzyme was highly stable at 90°C and retained 63% activity after heat treatment at 100°C for 1 h.