The organic residues in a glass bottle excavated from the Grand Baoen Temple site in the Song Dynasty, in Jiangsu Province, were studied in this paper. The Fourier transform infrared (FT-IR) spectroscopy, Gas chromatography–quadrupole mass spectrometry (GC-QMS) and Gas chromatography quadrupole time-of-flight mass spectrometry (GC-QTOF-MS) were applied to characterize the material. By comparing the 7 modern collected ambergris reference samples, the residue in the bottle was identified as ambergris. The biomarker molecule ambrein was detected in the residues via solvent vent mode and the co-eluent of interferent was checked using secondary Q-TOF mode. Furthermore, the occurrence of ambergris in the underground palace demonstrates the well-developed overseas incense trade and the solemn Buddhist culture of using incense in the Song Dynasty.