There is a global need for methods allowing rapid detection of pathogens in food samples, particularly for methods amenable for use in biosensors. Although antibodies have traditionally been applied for this purpose, the use of aptamers has been recognized as a promising alternative approach. Aptamers have many advantages, such as stability, low cost of production, and ease of modification. To identify DNA aptamers demonstrating binding specificity to Salmonella typhimurium, we applied rapid and simple whole-cell systematic evolution of ligands by an exponential enrichment (SELEX) method to an ssDNA library. FAM-labeled aptamers with high binding affinity to S. typhimurium, as determined by fluorescence spectroscopic analysis, were identified, and 1 aptamer (S6) with high binding affinity and specificity for S. typhimurium was selected via a process that required less than 3 months. In addition, by employing aptamer S6 in a nanogold-based colorimetric method, S. typhimurium could be detected at a concentration of 10⁶ CFU mL⁻¹. In this assay, the aptamer showed good selectivity for S. typhimurium. Thus, our whole-cell SELEX approach shortens the complex process required for identifying S. typhimurium specifically, rapidly, easily, and cost-effectively.