An automated procedure of sample preparation using pressurized liquid extraction (PLE) was developed for subsequent analysis by enzyme-linked immunosorbent assay (ELISA) for dioxins detection in ambient air samples collected from Burlington Ontario. Ambient air samples were collected from particle-phase using glass fibre filters (GFF) and from gas-phase using polyurethane foam from November 2014 to February 2015. The PLE extracts were cleaned up with acid silica followed by carbon mini-column. The average concentration of dioxins in particle phase was found to be 9.96±4.5 fgTEQ/m3 (n=10). This empirical finding is in agreement with high resolution gas chromatography –high resolution mass spectrometry (GC-MS) mean result of 10.04±2.9 fgTEQ/m3 (n=5). However, due to the limited sample size correlation between the two methods cannot be statistically established. The higher concentration of dioxins in Burlington, a city with heavy industry, was expected comparing the finding from previous study for downtown metropolitan Toronto (7.6 ± 2.0 fg BEQ/m3). Development of this method relied on calibration test, recovery test and Certified Reference Material (CRM) evaluation. Calibration test was successful in terms of developing standard curve with results within one standard deviation of the mean concentration of calibration standards. ELISA result on CRM was acceptable. Recovery test on extended toluene evaporation to half an hour or higher increased the recovery from 45% to an average of 82.4% for high concentrations and 89% for medium concentration of dioxins spike. The results of this study illustrate that PLE / ELISA can substitute for GC-HRMS as a cost effective screening tool to determine the dioxins concentration in ambient air.