Abstract. Anastasia L, Kodrat EA, Victor H, Sanjaya A, Pinontoan R. 2022. Isolation and characterization of indigenous amylolytic enzyme-producing Aspergillus sp. from sweet-flavored tapai. Biodiversitas 23: 5580-5586. Aspergillus sp. are able to produce abundant amylolytic enzymes that may have a crucial role in producing the sweet flavor of tapai. This study was aimed to evaluate the amylolytic activity of Aspergillus sp. isolated from a widely known sweet tapai from Jember, East Java, Indonesia. The fungi isolated from sweet tapai were identified under notable morphological parameters, and its amylolytic activities were evaluated by utilizing various starch-based media, such as potato peel, potato dextrose broth, cassava, and maize. Optimum pH and temperature of partially purified amylolytic enzymes were evaluated using DNS assay. The result of this study suggested that TM3 fungal isolate isolated from sweet tapai belonged to Aspergillus sp. section Nigri. Aspergillus sp. TM3 grown in the potato peel medium displayed the highest amylolytic activity, followed by those grown in maize, potato dextrose broth, and cassava media. Further characterization of partially purified amylolytic enzymes from the isolate showed that optimal pH for the amylolytic activity was at pH 4.59 while the optimal temperature was around 54°C. Furthermore, analysis by thin-layer chromatography revealed that the amylolytic enzymes starch hydrolysis yielded dextrose as the main product and oligosaccharides as the side product, which indicated that Aspergillus sp. TM3 may have both alpha-amylase and glucoamylase involved in its amylolytic activity. This study explored the role of Aspergillus sp. TM3 in producing the sweet flavor of tapai.