Many deadly sugar glider diseases remain underdiagnosed. Thus, this study aimed to detect the presence of Blastocystis sp. in sugar gliders (Petaurus breviceps). Fresh stools were taken from 100 3-month to 4-year old male and female sugar gliders from enthusiasts and breeders. Samples were directly observed in wet mount, stained with iodine, methylene blue, and giemsa, and cultured on simple and RPMI 1640 media. Results showed high detection of the parasite: 87% on wet mount, 94% on iodine staining, and 100% on methylene blue, giemsa staining and cultured media. Blastocystis sp. in sugar glider can be described as vacuolar, granular, cyst, and amoeboid, wherein vacuolar form predominated with size 0.38–2.95 µm (average of 1.46 µm). The parasite lived for 6 days in simple culture medium and 5 days on RPMI 1640 medium. Growth peak was marked on the third day for both media. This study is the first to report Blastocystis sp. in sugar gliders, revealing its presence in both fresh and cultured sugar glider stools.