Emissions of N2O from agricultural soils are an important source for this greenhouse gas. The present work examines the potential of Fourier transformed infrared (FTIR) spectroscopy coupled with a long path (LP) infrared (IR) gas cell for on-line measurement of concentration and isotopic signature of N2O emitted from soils. Nitrous Oxide was spectrally monitored during incubations of soil samples in a closed system under different conditions. Its emission from a Grumosol (Vertisol) was measured in presence and absence of acetylene, with various additions of nitrate and glucose, under aerobic and anaerobic conditions, and for two soil thickness layers. For comparison N2O emissions from a Terra Rossa (Cambisol) and a Hamra (Luvisol) were measured in the presence and absence of acetylene. In an additional experimental set the isotopic signature of emitted N2O was quantified after enrichment with K15NO3. Acetylene addition led to an increase in N2O emissions in all three soils but at various extents. Under aerobic conditions, N2O emission from the Grumosol became detectable only when running the experiments with a thicker soil layer (10 mm), suggesting the existence of coupled nitrification–denitrification. Nitrate addition to soils enhanced N2O emissions especially when coupled with glucose addition. Addition of15NO3− to the Grumosol resulted in the emission of all four N2O isotopologues: 14N2O,15N2O, 14N15NO, and 15N14NO. The observed slight delay in appearance of the species containing 15N and the relatively lower 15N enrichment of the N2O compared with the soil nitrate, indicate isotopic fractionation during denitrification. Yet, within the accuracy of our isotopic analysis, temporal emission patterns of 14N15NO and 15N14NO were similar indicating low possibility for “site preference” under the specific experimental conditions.