The disease caused by the opportunistic pathogen Vibrio harveyi is a serious infection which brings a heavy damage to aquaculture. Accurate and rapid detection of the microorganism is the first and necessary step to control the disease. Aptamers have a good potential application in the detection and identification of microorganisms because of their strong affinity, high specificity and good stability. In the present paper, aptamers targeting to V. harveyi were selected by the technique of systematic evolution of ligands by exponential enrichment (SELEX), and their affinities and specificities to V. harveyi were also evaluated. After 15 rounds of selection, the affinity of the aptamers in the enriched pool increased to 58.95, which was 16.8 times of that (3.51) in the original pool. After cloning and sequencing, 52 supposed aptamers were obtained from the enriched pool. These aptamers were divided into 8 families based on the homology analysis. Over 50% of these aptamers in the first and the second families shared higher homology, suggesting the selection was convergent and efficient. Six high-frequency aptamers were also found to have significant affinities and specificities to V. harveyi (P < 0.01). Five of them (S1,S25, S26, S27, S35) were proved to have higher affinities, and their affinity constants (K_d) were as followed: (32.6±7.1), (45.3±10.1), (24.7±5.8), (34.8±5.6) and (12.9±4.0) nmol/L, respectively. The production mechanism and the application values of high-frequency aptamers were also discussed. The present paper was the first report of aptamers with high affinity and specificity to the target V. harveyi, which would lay the foundation for the detection of the pathogenic microorganism based on aptamers.