The Ciliwung River in Jakarta urban areas has been degraded, emphasizing the importance of eDNA technology in producing additional biodiversity data. This study aims to analyze the current status of biodiversity in the Lower Ciliwung River, Jakarta. A total of 1,492,975 original reads were generated from nine samples collected at three sites. The sequences refer to 22, 24, and 28 classes at sites 1, 2, and 3, respectively. At the class level, the taxonomic composition was dominated by Coscinodiscophyceae, with a higher proportion (38.3%) than Insects (13.8%), Monogononta (10.8%), Mammalia (9.64%), Mamiellophyceae (7.27%), and Polychaeta (4.52%). With eDNA, we detect aquatic organisms, including fish, plankton, benthos, bacteria, and a large diversity of terrestrial and flying vertebrates. The biodiversity level was moderate, while the dominance level was categorized as high. The Reads sequence detected from eDNA water samples varied significantly between the three sites (ANOSIM-R = 0.43, p-value < 0.05). Most biota found have a high tolerance for pollutants and toxic materials. In general, the diversity is decreasing towards the lower reaches of the Ciliwung River. Our results indicate that eDNA could become an essential tool for biodiversity monitoring in a freshwater ecosystem.