Two-dimensional gel electrophoresis method for analysis of total proteins from Streptomyces roseolus was established in this paper as follows:IPG strips with pH 4-7, 2D clean-up kit purification protocol, 80 μg protein sample volume and silver staining. 2D-gels were obtained with low background, high resolution, good reproducibility and without obvious stripes. The 2-DE maps of total proteins from S. roseolus by induction of chitosan were analyzed by ImageMaster2D Platinum7.0, which showed 723±14 clearly visible protein spots. Compared with 2-DE maps of total proteins from S. roseolus incubated with no induction, 18 proteins detected in induced strain were in differential expression. Among these, 14 proteins were up-regulated and 4 proteins were down-regulated. 10 proteins whose expression levels differed more than 5 times were selected and identified by mass spectrometry. The results showed that the expressions of proteins such as elongation factor Tu, RNA polymerase, chaperone and so on were increased when incubating with chitosan, which suggested that they may be involved in the process of chitosan metabolism in S. roseolus.