Abstract: Assessment of urban human comfort is one of hot topics in the field of climate change. Due to rapid urbanization in developing countries, climate issues have gained increasing attention in cities. However, most of these studies are carried out in spatial and temporal variations of human comfort, few considers the effects of urbanization on human comfort. Therefore, the meteorological and urbanization data of Shanghai, Nanjing, Hangzhou and Hefei in the Yangtze River Delta are used to investigate the relationship between urbanization and human comfort, and determine the main urban factors on human comfort. First, the human comfort of four cities in the Yangtze River Delta during 1981—2010 is investigated, and the trends in cold or hot uncomfortable days of four cities are investigated, separately. The results show that Nanjing has the most uncomfortable cold days, while Hangzhou has the least, and Hangzhou has the most uncomfortable hot days while Nanjing has the least. There are downward trends in cold uncomfortable days, the decreasing tendency from large to small is Shanghai, Nanjing, Hangzhou and Hefei. There are upward trends in hot uncomfortable days, and the increasing tendency in descending order is Shanghai, Hangzhou, Nanjing and Hefei. Second, the levels of urbanization of four cities during 1991—2011 are analyzed by the fuzzy comprehensive evaluation, and the relationship between tendency of human comfort and the levels of urbanization are analyzed by the correlation method. The results show that there is a highly significant correlation between the levels of urbanization and the tendency rate of cold uncomfortable days, while no significant correlation is found between levels of urbanization and the tendency rate of hot uncomfortable days. Third, the urban factors on human comfort are investigated by gray relational analysis to determine main urban factors which affect human comfort. It is found that the main urban factor on human comfort is total population, while other more important factors are built-up area, total power consumption, public transportation vehicles, and capita green area.These results suggest that the urbanization of big cities reduce the number of cold uncomfortable days significantly. Urbanization, especially the factors of population increase, city expansion, and traffic congestion energy consumption have profound influences on human comfort of urban residents.