Understanding the occurrence, sources, transfer mechanisms, fugacity, and ecotoxicological risks of antibiotics play a pivotal role in improving the sustainability and ecological health of freshwater ecosystems. Therefore, in order to determine the levels of antibiotics, water and sediment samples were collected from multiple Eastern freshwater ecosystems (EFEs) of China, including Luoma Lake (LML), Yuqiao Reservoir (YQR), Songhua Lake (SHL), Dahuofang Reservoir (DHR), and Xiaoxingkai Lake (XKL), and were analyzed using Ultra Performance Liquid Chromatography/Tandem Mass Spectrometry (UPLC-MS/MS). EFEs regions are particularly interesting due to higher urban density, industrialization, and diverse land use in China. The findings revealed that a collective total of 15 antibiotics categorized into four families, which included sulfonamides (SAs), fluoroquinolones (FQs), tetracyclines (TCs), and macrolides (MLs), exhibited high detection frequencies, indicating widespread antibiotic contamination. The pollution levels in the water phase were in the order of LML > DHR > XKL > SHL > YQR. The sum concentration of individual antibiotics for each water body ranged from not detected (ND) to 57.48 ng/L (LML), ND to 12.25 ng/L (YQR), ND to 57.7 ng/L (SHL), ND to 40.50 ng/L (DHR), and ND to 26.30 ng/L (XKL) in the water phase. Similarly, in the sediment phase, the sum concentration of individual antibiotics ranged from ND to 15.35 ng/g, ND to 198.75 ng/g, ND to 1233.34 ng/g, ND to 388.44 ng/g, and ND to 862.19 ng/g, for LML, YQR, SHL, DHR, and XKL, respectively. Interphase fugacity (ffsw) and partition coefficient (Kd) indicated dominant resuspension of antibiotics from sediment to water, causing secondary pollution in EFEs. Two groups of antibiotics, namely MLs (erythromycin, azithromycin, and roxithromycin) and FQs (ofloxacin and enrofloxacin), showed a medium-high level of adsorption tendency on sediment. Source modeling (PMF5.0) identified wastewater treatment plants, sewage, hospitals, aquaculture, and agriculture as the major antibiotic pollution sources in EFEs, contributing between 6% and 80% to different aquatic bodies. Finally, the ecological risk posed by antibiotics ranged from medium to high in EFEs. This study offers valuable insights into the levels, transfer mechanisms, and risks associated with antibiotics in EFEs, enabling the formulation of large-scale policies for pollution control.