Objective Vibrio anguillarum (V. anguillarum) is an important conditional pathogenic bacterium, which can infect many aquacultural animals, and cause huge losses to aquaculture industry every year. Study on the pathogenic mechanism of V. anguillarum and rapid detection of the bacterium are necessary for the prevention of the disease. Aptamers show good application potential in many fields such as target analysis, detection of bacterium and research on pathogenic mechanism due to their high affinities and specificities. Therefore, selection of the aptamers against V. anguillarum and analysis of the related sites of the pathogen by its aptamers, can not only provide a new way for the identification of V. anguillarum, but also have important significance to explore the role of those sites in the disease control. Methods Aptamers against V. anguillarum were selected from the high frequency sequences by SELEX with sequencing in each selection round. Affinity of aptamer was measured by the ssDNA concentration method and the affinity and specificity of aptamers against the pathogen were also studied based on the measurement. The affinity constant (K_d) and maximum affinity (A_m) of aptamer were obtained by the nonlinear fitting according to the hyperbola function of the software Origin. Binding protein of aptamer H5 was isolated by magnetic separation and purified by polypropylene acyl amine gel electrophoresis (PAGE). The binding protein was identified by mass spectrometry. Its spatial structures and subcellular location were analyzed online by the websites of Prabi, Phyre2 and Psortb 3.0. Results An efficient selection method for aptamers was established based on each round of sequencing and high frequency sequences. A series of aptamers (H1, H5, H6, H12, H25, H26, H28, H33, H38 and H42) with good affinities and specificities towards the target bacterium V. anguillarum were selected by the efficient method. The K_d and A_m of 6 aptamers (H1, H5, H25, H26, H33, H38) were measured, and their K_d were (78.77±10.99), (180.65±23.01), (121.14±21.43), (276.42±51.23), (89.24±10.84), (167.12±23.73) nmol/L, respectively, and their A_m were (229.4±8.35), (891.04±50.14), (647.20±41.51), (720.85±75.35), (510.65±33.89), (576.06±38.73) nmol/L, respectively. The binding protein of aptamer H5 was identified as E1 component of pyruvate dehydrogenase in the cytoplasm of V. anguillarium. The main skeleton of the binding protein was composed of α-helix and β-fold, and the loops of random coil were mostly distributed outside of the protein. The interaction regions of the aptamer and its binding protein were also analyzed and speculated. Conclusion It was proved that the selection method based on each round of sequencing and high frequency sequences was quite efficient. Apparent affinity of aptamer was dependent on both K_d and A_m, and the A_m also played an important role in aptamer’s apparent affinity. Aptamer H5 entered into V. anguillarum probably by endocytosis, and then bound to E1 component of pyruvate dehydrogenase in the cytoplasm. The present study proved that aptamers could enter bacteria and bind to the corresponding targets, which provides a new idea for prevention of the disease caused by V. anguillarum and for development of novel aptamer medicines.