The secondary structure of silk fibroin (SF) and the compatibility of the two components were associated with the carboxymethyl chitosan (CMCS) content in SF/CMCS blend films. A rather complete conformation transition of SF from random coil to beta-sheet occurred after adding 5% CMCS into the SF film, and the blend film exhibited a high crystallinity and a good compatibility. The conformation of SF changed from beta-sheet to alpha-helix when blending 10% CMCS. When the CMCS content was up to 15%, the conformation of SF in blend films showed a transformation from beta-sheet to a random coil and their crystallinities decreased evidently; accordingly, there was a two-phase separation structure for the blend films containing 15% CMCS or more. However, the intermolecular interaction between the two polymers still existed in blend film with 15% CMCS or more. The blend films with 5-10% CMCS content showed the lower moisture absorption, swelling capacity, and solubility in water. These properties of blend films increased when adding CMCS more than 15%.