A pyrimidine-based sensor () has been synthesized through the facile one pot reaction of pyrrole-2-carboxaldehyde, 2-aminobenzimidazole and 1, 3-dimethylbarbituric acid in methanol using a few drops of HCl as a catalyst. Further, compound was fabricated into its fluorescent organic nanoparticles (FONPs) and characterized using DLS and SEM. The photophysical evaluation of the FONPs was done against bacteria and fungi. It was observed that the fluorescence intensity of the FONPs was selectively enhanced in the presence of the Gram -ve bacterium Pseudomonas aeruginosa with a detection limit of 46 CFU. To the best of our knowledge, this study is the first report on the use of the FONPs of pyrimidine derivative for the detection of Pseudomonas aeruginosa in various samples.