The fluorescence emission phenomena of polyamidoamine (PAMAM) have been discovered and characterized in recent decades. The amide, primary amine, and tertiary amine groups are present in PAMAM, which are not the traditional or typical fluorescence emission groups. The fluorescence emission groups or mechanism of PAMAM were yet not clear but cause cares. In this study, PAMAM was characterized using NMR (¹⁵N NMR, ¹³C NMR, ¹H NMR, and N–H 2D NMR), IR, and MS. The results proved that amide resonance structures, corresponding to imidic acid and tertiary ammonium groups, existed in PAMAM. The new imidic acid and tertiary ammonium groups found in PAMAM might help explain the intrinsic-fluorescence phenomena.