The Tibetan Tripitaka is a collection of the Tibetan versions of Indian Buddhist classics and writings, and is of high historical, scientific and artistic value. In this work, optical microscopy (OM), scanning electron microscopy-energy dispersive X-ray spectrometry (SEM-EDX), Raman spectrometry, high performance liquid chromatography-mass spectrometry (HPLC-MS), microbial purification & cultivation technology, molecular biology identification technology and high-throughput sequencing method were applied to a sample (S1) Tibetan Tripitaka paper with gold writing and 2 samples (B1 and B2) coated with microorganisms of the Ming and Qing Dynasties. It was found that 1) the gold writing material is particles of a gold-silver-copper alloy containing Au (72.8%—75.6%), Ag (22.8%—26.0%) and Cu (1.2%—1.6%), indicating a relatively low purity of gold mud used; 2) paper was dyed with indigo, coated with graphite; 3) pH value is 7.0—7.5, suggesting that it has not been acidified and 4) a large number of fungal spores scattered among paper fibers. The microbial secretions are one of the main causes of paper adhesion during the preservation. The results of microbial purification and cultivation and molecular biology identification show that the green mold on the surface of Tripitaka Sample B2 is Aspergillus fumigatus. The high-throughput sequencing results show that the microbial species attached to the surface of Tripitaka Sample B1 included Chaetomium sp., Aspergillus sp., Mucor sp. and Beauveria sp., etc.