Pseudomonas aeruginosa is a pathogen of critical priority importance according to the WHO. Due to its multi-resistance and expression of various virulence factors, it is the causal agent of severe healthcare-acquired infections (HAIs). Effective strategies to control infections caused by P. aeruginosa must include early and specific detection of the pathogen for early and timely antibiotic prescription. The need to develop a specific and reproducible diagnostic technique is urgent, which must often be more sensitive and faster than current clinical diagnostic methods. In this study, we implement and standardize the loop-mediated isothermal amplification (LAMP) technique, coupled with the reverse line blot hybridization (RLBH) technique for the detection of P. aeruginosa. A set of primers and probes was designed to amplify a specific region of the P. aeruginosa 16s rRNA gene. The sensitivity of the LAMP-RLBH method was 3 × 10⁻⁴ ng/μL, 1000 times more sensitive than the PCR and LAMP technique (this work), with a sensitivity of 3 × 10⁻³ ng/μL. The LAMP-RLBH and LAMP techniques showed specific amplification and no cross-reaction with members of the ESKAPE group and other Pseudomonas species. The present investigation provides a technique that can be easily performed in less time, achieving a faster and more reliable alternative compared to traditional microbial diagnostic methods for the detection of P. aeruginosa.