Pseudomonas aeruginosa is an opportunistic pathogen that is ubiquitously distributed throughout the environment, particularly in moist habitats. It causes sever economic losses in poultry industry. It also causes serious diseases in human especially in nosocomial patients lead to high death rates. The main objective in this study is to explain the prevalence rates of P. aeruginosa in broilers and human. So, the samples were collected from 100 broiler chicken at different age groups; one day old chicks n=50, 14 days old chickens n=25 and 28 days old chickens n=25. From each chick, different organs were collected (liver, gall bladder, intestine, lungs, cloacal swabs, n=100 each). Yolk sac samples were collected only from one day old chicks (n=50).Thirty samples were collected from human: urine samples from patients complain from urinary tract infections (UTIs) (n=17) and pus swabs from abscesses (n=13) at Qalyubia Governorate , Egypt. The results of bacteriological examinations revealed that P. aeruginosa was recovered from; 47/100 chickens (47%), 5/17 (29.41%) urine samples, and 3/13 (23.07%) pus swabs. A Total of 55 isolates (47 isolates from broilers and 8 isolates from human) were identified by MALDI-TOF MS. It concluded that MALDI-TOF MS identification were 24/55 (43.63%), while by conventional method were 55/100 (55%) ; ( from broilers and human) , so MALDI-TOF MS is more accurate than conventional method.