Atmospheric carbon dioxide (CO2) concentrations observed in urban areas and the surroundings often show short-term elevations on a time scale ranging from several hours to a few days. These variations are considered to be attributed to the CO2 emissions from biotic activities and burning of fossil fuels. Partitioning the contribution rate between the individual CO2 sources would be useful to reduce the uncertainty of the CO2 emissions estimated from atmospheric inversion calculations. The ratio of oxygen (O2) consumption to CO2 emission for fossil fuel burning (−O2/CO2) ranges from 1 to 2, depending on the elemental composition of the fossil fuel. The average −O2/CO2 exchange ratio for the fossil fuel consumed in Japan is more than 1.4 while that for land biotic process is 1.1. Therefore, the difference in the exchange ratios could allow us to determine the contributions from the individual sources. In this study, we started a continuous observation of the atmospheric CO2 and O2 concentration at Tsukuba in February 2015, and evaluated the individual contributions of the biotic and fossil fuel-derived emissions to the short-term CO2 variations based on the observed −ΔO2/ΔCO2 changing ratios. The observed −ΔO2/ΔCO2 changing ratios show clear seasonal variation with minimum in summer and maximum in winter, ranging from 1.0 to 1.6. Taking into account of the average −O2/CO2 exchange ratio of 1.58 for the fossil fuel consumption in Tsukuba, we obtained the contribution ratios of fossil fuel burning to the CO2 variations were more than 70% in winter and 0–30% in summer. To validate the above estimations based on the −O2/CO2 exchange ratio, we conducted flask samplings of air 6 times at the different months during the observation period and measure 14CO2, which is known as good indicator of the burning of fossil fuels. Note that 5 flask samples were usually collected for each sampling at an interval of 3 hours to detect the diurnal variations. The contribution rates of the fossil fuel CO2 estimated from −O2/CO2 ratios and 14CO2 agree each other within the uncertainties.