Fungal spores constitute the most abundant fraction of biological aerosols in the atmosphere, influencing human health, the biosphere, atmospheric chemistry, and climate. However, the total abundance of fungal spores in the atmosphere is rather uncertain and likely underestimated to a large extent by traditional Colony Forming Units (CFU) assays. In this study, flow cytometry (FCM) was utilized in combination with fluorescent stains for the rapid counting of ambient fungal spores, with complementary quantification of two molecular tracers for fungal spores. The FCM results had significant positive correlation with the concentrations of the fungal tracers (R2 was 0.75 and 0.70 for arabitol and mannitol, respectively). During this study, total particle counts, fungal spore numbers and the fractions of fungal spores of the total particle numbers were in the range of 44,698–9,54,211 m−3, 8224–261,154 m−3 and 1.9–46.5%, respectively, at an urban location in northern China. Meteorological conditions were shown to have complex effects on the ambient concentrations of fungal spores: the number concentrations of fungal spores exhibited significant positive correlation with relative humidity and temperature, negative correlation with wind speed and no relationship with solar radiation during the sampling period.