Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic and nosocomial bacterial pathogen. Various multi-resistance mechanisms present across numerous P. aeruginosa strains counteract conventional antimicrobial therapy, thereby becoming a great challenge. This study aimed to establish the application of immunomagnetic isolation and chemiluminescence to detect the presence of extended spectra of β-lactamases encoding genes: blaTEM and blaVEB; metallo-beta-lactamases encoding gene: blaVIM; aminoglycoside modifying enzymes encoding gene: aac(6)II, ant(3)I; and the specific gene for P. aeruginosa, gyrB. P. aeruginosa was specifically selected using the immunomagnetic nanoparticles (IMNPs) in the six parallel bacterial plates counting, proving that they are reliable. Then, the high efficiency of IMNPs@Probes in targeting the resistance genes of P. aeruginosa was demonstrated using the results of chemiluminescent intensities of blaTEM, blaVEB, blaVIM aac(6)II, ant(3)I, and gyrB (more than 10 times higher than that of the control). Sixty-eight in situ clinical samples were tested for the presence of these resistance genes, and one more blaTEM and three more blaVIM individuals were detected using this method compared to the traditional PCR. Thus, the application of our method in clinical screening is specific, accurate, and reliable, and it could be useful in the administration of appropriate treatment.