This study reports the transformation of nitrogen during liquefaction of two microalgae (i.e., Chlorella vulgaris and Spirulina) in subcritical/supercritical ethanol at 220−260 °C. The nitrogen contents in all products from microalgae liquefaction were quantified, and the nitrogen species in those products were characterized. The nitrogen in the raw microalgae are mainly present in the form of protein-N, with a small fraction of nitrogen present in other forms such as quaternary-N, inorganic-N, and pyridinic-N/amine-N. The nitrogen in the solid residue is found to decrease significantly at 220 °C. As the liquefaction temperature further increases, the nitrogen in char gradually decreases, accompanied by the increase of nitrogen in bio-oil, i.e., up to 83.2% at 260 °C for Chlorella vulgaris and 90.1% at 250 °C for Spirulina. The results demonstrate that the nitrogen species in the raw microalgae are not stable and easily transformed into other N-containing species in bio-oil (mainly the pyrrole and amine species). A small fraction of pyrrolic-N is also formed in the solid residue at 220 °C, while the formation of N-containing gas species is negligible.