The effects of gel media, sample application and elution flowrate on the activity recovery and aggregation in refolding of bovine carbonic anhydrase B (CAB) by size exclusion chromatography (SEC) were investigated. Variation in aggregation was demonstrated visibly by the comparison of refolding profiles under different operating conditions. Some principles with regard to practical application were proposed. Meanwhile, the analysis of relationship between peak resolution and activity recovery provided evidences for the mechanism of size exclusion chromatography protein refolding.