Abstract: Abstract: Collagen, as the most abundant structural protein in living bodies, constitutes approximately 30% of the total proteins. Collagen exhibits excellent film-forming property, and can be used be as an outstanding carrier of bioactive compounds (antioxidant and antimicrobial) for food packaging. However, the rough surface and defects in mechanical properties limit application of collagen film for food packaging. Chitosan, industrially produced by partial deacetylation of chitin, is also one kind of excellent natural film-forming material. The incorporation of chitosan can increase the antimicrobial ability of packaging film. Chitosan-gelatin blend films show better material properties when compared to pure single-component polymer films. The previous studies inferred hydrogen bonds and electrostatic interactions were formed between collagen and chitosan molecules during the process of film formation using one-dimensional infrared spectroscopy. Two-dimensional infrared correlation spectroscopy is a method to study the intermolecular interactions between functional groups under the interference of exogenous factors. The two-dimensional infrared correlation spectroscopy can also obtain the sensitivity and response order of the functional groups to external disturbance. At present, the interaction between bone collagen and chitosan has not been reported by two-dimensional infrared correlation spectroscopy. The paper aimed to infer the interaction by analyzing the characteristics of two-dimensional infrared correlation spectroscopy of bone collagen-chitosan blend film with different mixing ratios. Collagen, extracted from sheep bone by pepsin, and chitosan were mixed evenly according to 100:0, 60:40, 50:50, 40:60 and 0:100, respectively, and then 25% glycerol was added as plasticizer. The mixed film-forming solution was dried at 50 oC for 18 h, and then placed at room temperature for 6 h and uncovered. Fourier transform infrared spectroscopy (FTIR) of blending film was obtained and analyzed by the peak fitting and two-dimensional correlation analysis to infer the interaction between collagen and chitosan molecules. Infrared spectroscopy showed that the intensity and shape of infrared absorption peaks changed obviously with the addition of chitosan. The results showed that conformation of C=O bond of bone collagen varied first with the addition of chitosan. The peak fitting results showed that the secondary structure of collagen in the blend film was mainly β sheet. The content of α helix decreased, and the content of β sheet and β turn increased after adding chitosan. The change of secondary structure of collagen in the blend film may be caused by the film forming process, such as the blending temperature and drying temperature. The secondary structure of collagen extended gradually, and further characteristic group was exposed. FTIR results proved the higher deacetylation degree of chitosan. The existence of two-dimensional infrared correlation peak involved in C-O-C bond of glycosyl skeleton confirmed the spatial conformation of chitosan varied. The infrared spectrum of N-H bond vibration varied significantly after adding chitosan. The hydroxyl, amine, and carboxyl groups of collagen were capable of forming hydrogen bonds with hydroxyl and amine groups of chitosan. The electrostatic interaction was formed between collagen and chitosan whose -NH2 groups were easily protonated in acidic solutions and converted to -NH3+. Two-dimensional infrared correlation spectroscopy indicated the response order of groups to mixture ratio was: 1633 cm-1 > 1448 cm-1 > 1236 cm-1 > 1068 cm-1 > 997 cm-1 > 896 cm-1. The intensity and maximum wavenumbers of absorption peaks confirmed that the intermolecular hydrogen bonds appeared between collagen and chitosan in blend films, which were dependent on the ratio of collagen/gelatin to chitosan. In conclusion, the present study shows that hydrogen bonding and electrostatic interaction are formed between collagen and chitosan molecules in blending film, and the strength of intermolecular interaction is related to mixture ratio of collagen and chitosan.