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High concentrations of ozone (O3) is a major air problem in urban areas, which creates a serious threat to human health. Urban street canyon morphology plays a key role in air pollutant dispersion and photochemical reaction rate. In this study, a one-year observation at three height levels was performed to investigate the O3 distribution vertically in a street canyon of Shenyang. Then, field investigation and ENVI-met modelling were conducted to quantify the influence of street canyon morphology and microclimatic factors on O3 distribution at the pedestrian level. All O3 concentrations at the three height levels were high from 1:00 p.m. to 4:00 p.m. Both O3 concentrations at pedestrian level and the middle level in the canyon were 40% higher than at roof level. O3 accumulated in the canyons rather than spread out. The in-canyon O3 concentrations had significantly positive correlations with building height, aspect ratio, sky view factor, air temperature, and wind speed. Both field investigation and ENVI-met modelling found high O3 concentrations in medium canyons. Photochemical reaction intensity played a more important role in in-canyon O3 distribution than dispersion. Wide canyons were favorable for removing O3.