Understanding the prevalence of airborne fungi in a city or region is important for ecological diagnosis and specific treatment of allergic manifestations induced by inhalation of fungal allergens. The present study was conducted to assess characteristics and variation of culturable airborne fungi at four selected sampling sites in Hangzhou, southeast China. Results showed that concentration of culturable fungi in the air ranged from < 12 colony forming units (CFU) m-3 to 8767 CFU m-3 with mean of 848 CFU m-3. We identified a total of 352 fungal isolates from multiple sampling sites and across seasons, which were distributed across 21 genera and 85 species of fungi. Penicillium, Cladosporium, Alternaria, Aspergillus, and Trichoderma were the most predominant fungi based on their frequency and concentration percentage, and fungal composition differed from site to site and from season to season. Approximately 36.5% of the total number of isolated fungal species belonged to Penicillium, which also represented the maximum proportion of the total fungal concentration at about 29.6%. The fungal species with higher frequency in Hangzhou were P. chrysogenum (7.7%), C. cladosporioides (6.3%), Alternaria alternata (5.6%), P. funiculosum (4.3%), Aspergillus sydowii (4.0%). Moreover, there was significant variation between sampling sites, with higher fungal concentrations detected at Zhejiang Gongshang University Jiaogong Campus (ZJGSUJC) and Breeze-ruffled Lotus at Quyuan Garden (BLQG), followed by Yan’an Road Business Street (YRBS), while the lowest concentrations (P < 0.05) were found at Tianmushan and Jiaogong Cross Road (TJCR). Furthermore, different patterns of seasonal variation in fungal concentrations were found at different sampling sites, and the mean fungal concentration at all four sites was lowest during the winter, while there was no difference among summer, autumn, and spring. Our results can provide a baseline for studying airborne culturable fungi in southeast China, and will enable evaluation of the risks to human health from exposure to the atmosphere.