Abstract: In order to explore the physical and chemical properties, spatial structure and antigenic sites of Ⅸ protein in Canine adenovirus type Ⅰ（CAdV-1）,in this study, the Ⅸ protein amino acid sequence of the virus that was isolated from the laboratory of the Research and Development Team of Pet Biological Products in Institute of Special Animal and Plant Sciences of Chinese Academy of Agricultural Sciences, were used as the research object. Bioinformatics methods were used, and online database websites such as ProtParam tool, NetPhos 3.1 Server, TMHMM,PSIPRED,SOPMA,Phyre2 and IEDB were used to predict physicochemical property, phosphorylation site, glycosylation site, transmembrane domain, signal peptide, secondary structure, tertiary structure, antigen epitope and B cell epitope of Ⅸ protein. The results showed that Ⅸ protein contained 71 amino acid with an average water absorption coefficient of-0.399 and isoelectric point of 9.86. The α-helix, extended chain, β-corner sum and random coil were 16.90%,35.21%,9.85%,and 38.03%,respectively. There was no transmembrane domain and signal peptide in the protein; there were 2 potential protein antigenic determinant, 5 protein phosphorylation sites, and the potential B cell antigenic determinant was positions 18-31. The results suggested that the preliminary prediction and analysis of physicochemical properties and antigenic determinants of CAdV-1 Ⅸ protein were obtained in this experiment, which could provide a reference for the isolation and purification of CAdV-1 Ⅸ protein, and a preliminary basis for further development of subunit vaccine based on Ⅸ protein.