Nitrous oxide (N2O) is a major radiative forcing and stratospheric ozone-depleting gas. Among natural sources, freshwater ecosystems are significant contributors to N2O. Although temperature is a key factor determining the N2O emissions, the respective effects of temperature on emitted and dissolved N2O in the water column of freshwater ecosystems remain unclear. In this study, 48 h incubation experiments were performed at three different temperatures; 15 °C, 25 °C, and 35 °C. For each sample, N2O emission, dissolved N2O in the overlying water and denitrification rates were measured, and N2O-related functional genes were quantified at regular intervals. The highest N2O emission was observed at an incubation of 35 °C, which was 1.5 to 2.1 factors higher than samples incubated at 25 °C and 15 °C. However, the highest level of dissolved N2O and estimated exchange flux of N2O were both observed at 25 °C and were both approximately 2 factors higher than those at 35 °C and 15 °C. The denitrification rates increased significantly during the incubation period, and samples at 25 °C and 35 °C exhibited much greater rates than those at 15 °C, which is in agreement with the N2O emission of the three incubation temperatures. The NO3− decreased in relation to the increase of N2O emissions, which confirms the dominant role of denitrification in N2O generation. Indeed, the nirK type denitrifier, which constitutes part of the denitrification process, dominated the nirS type involved in N2O generation, and the nosZ II type N2O reducer was more abundant than the nosZ I type. The results of the current study indicate that higher temperatures (35 °C) result in higher N2O emissions, but incubation at moderate temperatures (25 °C) causes higher levels of dissolved N2O, which represent a potential source of N2O emissions from freshwater ecosystems.