The use of synthetic N fertilizers has grown exponentially over the last century, with severe environmental consequences. Most of the reactive N will ultimately be removed by denitrification, but estimates of denitrification are highly uncertain due to methodical constraints of existing methods. Here we present a novel, mobile isotope ratio mass spectrometer system (Field-IRMS) for in-situ quantification of N and NO fluxes from fertilized cropping systems. The system was tested in a sugarcane field continuously monitoring N and NO fluxes for 7 days following fertilization using a fully automated measuring cycle. The detection limit of the Field-IRMS proved to be highly sensitive for N (54 g ha day) and NO (0.25 g ha day) emissions. The main product of denitrification was N with total denitrification losses of up to 1.3 kg N ha day. These losses demonstrate sugarcane systems in Australia are a hotspot for denitrification where high emissions of NO and N can be expected. The new Field-IRMS allows for the direct and highly sensitive detection of N and NO fluxes in real time at a high temporal resolution, which will help to improve our quantitative understanding of denitrification in fertilized cropping systems.