Avian Pathogenic Escherichia coli (APEC) is one of the most important bacterial pathogens of poultry. The lack of suitable vaccines and the emergence of multi-resistant strains have hampered the control of avian colibacillosis. To identify immunogenic proteins of APEC as vaccine candidates, immunoproteomics and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) were applied. Proteins from total cell lysates of APEC DE205B isolated from the brain of a duck with septicemia and neurological symptom in China were separated by two-dimensional electrophoresis (2-DE) and reacted with hyperimmune duck serum against DE205B. Fourteen immunoreactive spots were found, representing 11 distinct proteins. These included two predominant immunogenic components, outer membrane protein A (OmpA) and flagellin (FliC). GroEL, which is a member of the molecular chaperone family and identical structurally to eukaryotic heat shock protein 60 (Hsp60), and the other eight antigens are reported here as immunoreactive proteins of APEC for the first time. Subsequently, nine genes encoding the identified proteins were successfully cloned and expressed in E. coli BL21 (DE3). Seven of the recombinant proteins were able to react with hyperimmune duck serum and three of them, GroEL, OmpA and FliC, showed stronger immunoreactivity. Challenge studies revealed that, just like OmpA and FliC, recombinant GroEL stimulated a strong antibody response and supported protective efficacy against APEC infection in ducks. With high phylogenetic conservation, it is considered that GroEL would be an ideal immunogen of APEC for vaccine development.