Abstract: Removal of protein in ε-polylysine (ε-PL) fermentation broth is an important step before the extraction of ε-PL.With the relative quality contents of ε-PL and protein as the measurement indexes, the research carried out a comparision between frozen processing and thermal treatment to remove protein and the result indicated that the latter was preferred.Then the thermal treatment conditions were optimized as pH6.0, temperature 80℃ and treat time 15min.Under this optimal conditions, removal rate of protein and retention rate of ε-PL reached 83% and almost 100% respectively.SDS-PAGE was employed to evaluate the effect of protein removal and the result showed that the molecular weight of protein in ε-PL fermentation broth distributed below 66400, only those whose weight below 14300 were hard to be removed by thermal treatment.Research on thermal treatment of ε-polylysine fermentation broth derived some significance.