Aim To separate and purify human recombinant apolipoprotein E (Apo E) from Escherichia coli strain BL21(DE3). Methods PET32a Apo E constructs were transformed into BL21 and protein expression was induced. Apo E thioredoxin fusion protein was separated by Ni 2+ affinity column. After digestion of thioredoxin by thrombin, Apo E was purified by Sephacryl S 300 gel filtration column. Results Apo E₂, Apo E3 and Apo E4 were produced by this method, with high yield and high purity. Conclusions A new system for separation and purification of human recombinant Apo E was successfully established.