A PCR-enzyme-linked immunosorbent assay (PCR-ELISA) was developed for direct identification of Pseudomonas aeruginosa from positive BACTEC blood culture bottles. PCR primers were designed to target a 249 bp sequence of the oprI gene in P. aeruginosa. Biotin-labeled probe (PA3) targeted to the species-specific motif were hybridized to the digoxigenin-labeled amplified products from P. aeruginosa and captured on streptavidin-coated microtiter plates. The specificity of the assay using the PA3 probe was investigated with a range of microorganisms, which are commonly isolated from blood culture bottles serving as negative controls. The PCR-ELISA assay was shown to be highly specific for the identification of P. aeruginosa and was 10-fold more sensitive than an agarose gel-based detection method using the same pair of primers, with a detection limit at 10 fg of template. The PCR-ELISA assay developed in this study is 100% sensitive and 100% specific as it correctly identified all 73 positive and 42 negative controls as well as 25 double blind clinical samples. It significantly reduces the time needed for the identification of P. aeruginosa from positive BACTEC blood cultures bottles from 2-3 days to 6-8h.