A total of 34 antibiotics from five major classes of antibiotics, including macrolides, sulfonamides, quinolones, tetracyclines and chloramphenicol, were considered as contaminants, considering the Yellow River Estuary as the study area. The distribution, sources and ecological risks of typical antibiotics in the Yellow River Estuary were investigated using an optimized solid-phase extraction pre-treatment and an Agilent 6410B tandem triple-quadrupole liquid chromatography–mass spectrometer for antibiotic detection. The results show that antibiotics were widely present in the water bodies of the Yellow River Estuary, with 14 antibiotics detected to varying degrees, including a high detection rate for lincomycin hydrochloride. Farming wastewater and domestic sewage were the primary sources of antibiotics in the Yellow River Estuary. The distribution characteristics of antibiotics in the study area were linked to the development of farming and social activities. The ecological risk evaluation of 14 antibiotics in the Yellow River Estuary watershed showed that clarithromycin and doxycycline hydrochloride were present at medium-risk levels, and lincomycin hydrochloride, sulfamethoxazole, methomyl, oxifloxacin, enrofloxacin, sulfadiazine, roxithromycin, sulfapyridine, sulfadiazine and ciprofloxacin were present at low-risk levels in the samples collected from water bodies of the Yellow River Estuary. This study provides novel, beneficial information for the assessment of the ecological risk presented by antibiotics in the Yellow River Estuary water bodies and provides a scientific basis for future antibiotic pollution control in the Yellow River Basin.