Pseudaminic acid (Pse) has been known for participating in crucial bacterial virulence and thus is an attractive target in the development of glycoconjugate vaccine. Particularly, this therapeutic alternative was suggested to be a potential solution against antibiotic resistant Acinetobacter baumannii that poses a serious global health threat. Also, Pse was found to be involved in the exopolysaccharide (EPS) of mild antibiotic resistant A. baumannii strain 54149 (Ab-54149) of which specific glycosyl linkage can be depolymerized by phage (Phi AB6 tailspike protein (Phi AB6TSP). In this study, we found that the antibodies induced by Ab-54149 EPS was capable of recognizing a range of EPS of other clinical A. baumannii strains, and deemed as a great potential material for vaccination. To efficiently acquire homogeneous EPS-derived oligosaccharide with significant immunogenic activity for the production of glycoconjugate, we used the Phi AB6TSP for the fragmentation of Ab-54149 EPS instead of chemical methods. Moreover, insight into the ligand binding characterization of Phi AB6TSP suggested the branched Pse on the Ab-54149 EPS served as a recognition site of Phi AB6TSP. The serum boosted by Phi AB6TSP-digested product and carrier protein CRM197 conjugate complex displayed specific sensitivity toward Ab-54149 EPS with bacterial killing activity. Strikingly, Pse is an ideal epitope with strong antigenicity, profiting the application of the probe for pathogen detection and glyco-based vaccine.