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Nitrogen dioxide (NO2) is one of the main pollutants that affecting the quality of life and human health. Mostly, the effects of NO2 exposure depending on concentration, duration of exposure and anthropometric variables. This study aimed to analyzing the NO2 concentration of ambient air and to assessing human health risks in real time and lifetime exposure. To achieve that goal, air samples were taken at four strategic locations in Kupang City, in which were represented by industrial, roadside, residential, and office areas. The Griess Saltzman method was used for sampling and analysis of NO2 levels. Totally, there were 48 air samples had been analyzed for NO2 levels. The detected NO2 concentration showed a trend varied between sampling locations. Overall, NO2 levels measured still met ambient air quality standards. Human health risk assessment was carried out by determining the intake (I) and Risk Quotient (RQs) in four groups of individuals exposed to NO2. The results shown that the RQs for real time and lifetime exposures were both less than 1.0, in which implying a non-carcinogenic risk of NO2 exposure in the study area. The results Health risk assessment also indicating that potential non-carcinogenic risks occur when NO2 exposure is more than 39 years.