Pseudomonas aeruginosa is a clinically significant pathogen with high antibiotic resistance, necessitating rapid and reliable diagnostic methods. In this study, we employed a whole-cell SELEX strategy to select ssDNA aptamers that specifically recognize P. aeruginosa. After 10 selection rounds, six aptamer candidates were obtained and evaluated for affinity. To demonstrate the diagnostic potential, aptamer T1 was integrated into a hybrid lateral flow immunoassay (LFIA), replacing the traditional detection antibody. In this assay, the AuNP–aptamer complex interacts with the target bacteria and is captured by a specific antibody immobilized on the test line. The LFA achieved a visual detection limit of ~102 CFU/mL within 15 minutes, demonstrating high specificity and potential for point-of-care applications. This study highlights the feasibility of using aptamers as low-cost, rapidly synthesized recognition elements in bacterial diagnostics and lays the groundwork for aptamer-based biosensor development for other infectious agents.