Urinary tract infection (UTI), which can be caused by various pathogens, if not detected at an early stage can be fatal. It is essential to identify the specific pathogen responsible for UTI for appropriate treatment. This study describes a generic approach to the fabrication of a prototype for the noninvasive detection of a specific pathogen using a tailor-made plasmonic aptamer-gold nanoparticle (AuNP) assay. The assay is advantageous because the adsorbed specific aptamers passivate the nanoparticle surfaces and reduce and/or eliminate false-positive responses to nontarget analytes. Based on the localized surface plasmon resonance (LSPR) phenomena of AuNP, a point-of-care aptasensor was designed that shows specific changes in the absorbance in the visible spectra in the presence of a target pathogen for robust and fast screening of UTI samples. In this study, we demonstrate the specific detection of Klebsiella pneumoniae bacteria with LoD as low as 3.4 × 103 CFU/mL.