Yersinia enterocolitica remains a threat to public health, and a sensitive detection method is a prerequisite due to its complicated diagnosis associated with slow growth. Recently, aptamer-based detection systems have played a vital role in the development of simple, rapid, sensitive, and specific detection methods. Herein, highly specific ssDNA aptamers were screened against Y. enterocolitica at the different growth stages by whole cell-SELEX. Cells at different growth stages were harvested and incubated with an ssDNA library to get an enriched pool of specific aptamer candidates. After the 10th round of SELEX, the enriched pool was sequenced and grouped into seven families based on homology and similarity of the secondary structure. Flow cytometry analysis revealed that the aptamers M1, M5, and M7 with Kd values of 37.93 ± 7.88 nM, 74.96 ± 21.34 nM, and 73.02 ± 18.76 nM had the highest affinity and specificity to the target, respectively. The selected aptamers showed binding to the different growth stages of Y. enterocolitica with a significant increase in the gated fluorescence. Our aptamer selection strategy is convenient, and the developed aptamer can be useful for an accurate and reliable detection system.