The ¹⁵N gas flux method can be used to determine the emission factor of N₂O being unnecessary the inclusion of treatments without the application of fertilizer. In Brazil, the world’s largest sugarcane producer, it is common the use of vinasse (liquid waste from ethanol production) as organic fertilizer for the crop, and this practice can increase N₂O emissions, because the vinasse is rich in carbon (C) and nitrogen (N). The aim of this study was to determine the emission factor of a sugarcane crop, ratoon cane, with or without the application of vinasse, using the ¹⁵N gas flux method. The study was conducted in São Paulo State, Brazil, using a randomized block design with a 2 x 2 factorial treatment containing four replicates and a total 16  plots with one 1.5-m long rows of sugarcane in 1.5m intervals. The factors corresponded to application or not of vinasse (100 m³ ha^-1) in plots with two different levels of N (55 and 110 kg ha^-1 as ammonium nitrate doubly labeled with ¹⁵N at 31.9 atom %). During 10 consecutive days, gas samples were taken from glass chambers (6 cm of diameter, 7 cm of height, 200 cm³) installed in the line of fertilization. The chambers were closed for 3 h (from 8 to 11 am) to allow N₂O detection. The concentration and ¹⁵N content of N₂O-N was determined by automated isotope ratio mass spectrometry (IRMS). Cumulatively, the highest emissions were observed for ratoon cane treated with vinasse. The emission factor ranged from 0.01 (without vinasse) to 0.35 % (with vinasse), both smaller than the 1% suggested by the Intergovernmental Panel on Climate Change. The ¹⁵N gas flux method allowed quantifying N₂O emissions under field conditions, indicating a low emission factor for the cultivation of sugarcane, even using vinasse.