In this work, we present a method for absolute measurement of air fluorescence yield based on high resolution optical emission spectroscopy. The absolute measurement of the air fluorescence yield is feasible using the Cherenkov light, emitted by an electron beam simultaneously with the fluorescence light, as a “standard candle”. The separation of these two radiations can be accomplished exploiting the “dark” spectral regions of the emission band systems of the molecular spectrum of nitrogen. In these “dark” regions the net Cherenkov light can be recorded experimentally and be compared with the calculated one. The instrumentation for obtaining the nitrogen molecular spectra in high resolution and the noninvasive method for monitoring the rotational temperature of the emission process are also described. For the experimental evaluation of the molecular spectra analysis we used DC normal glow discharges in air performed in an appropriate spectral lamp considered as an air-fluorescence light emulator. The proposed method and the associated instrumentation could be tested and used in thin or thick target experiments in electron beam accelerators as a candidate optical system for this purpose.