This study presents a novel multiplex PCR technique designed to identify adulteration in refined edible bird's nests (EBNs), specifically targeting common adulterants such as pig skin and Sterculia gum. Distinctive PCR product sizes of 294 bp for pig DNA and 157 bp for EBN DNA, together with 750 bp for Sterculia gum were achieved, demonstrating a sensitivity of 1 ng/μL for the three of the samples, which underscores the method's specificity. Optimal conditions were established with a primer concentration of 20 μM and an annealing temperature of 59.5ºC for pig skin detection, while for Sterculia gum, a primer concentration of 10 μM and an annealing temperature of 57ºC were effective using ITS and COI primers, respectively. This method offers unprecedented precision in distinguishing between adulterants in purified EBNs, providing a significant advancement in ensuring the integrity and safety of these products in the field of oriental medicine and cuisine.