As a severe intestinal disorder, short bowel syndrome (SBS) has become the leading cause of chronic intestinal failure worldwide. Teduglutide, a 33-amino-acid glucagon-like peptide-2 (GLP-2) analog, offers a novel therapeutic option for individuals suffering from SBS. In this study, Teduglutide purity certified reference material (CRM) with certified value and expanded uncertainty of (85.0% ± 2.7%) was developed with Teduglutide produced through recombinant gene expression technique. After a comprehensive purity verification and structure confirmation, two independent isotope dilution mass spectrometry (IDMS) strategies based on amino acid (AA) and sulfur measurements were employed for CRM certification, with the later strategy being used for the first time to certify a solid purity CRM. The Teduglutide purity CRM showed good homogeneity, it also exhibited satisfying stability for at least 14 days at 4 °C, 24 h for 25 °C and 12 months at -20 °C. The development of Teduglutide purity CRM (GBW09342) enables the establishment of accurate and reliable analytical methods for purity assessment, which is essential for ensuring the quality, consistency, and clinical safety of corresponding pharmaceutical products. It also provides a reliable reference for the quality control of other peptide-related drugs.