Organotin compounds are man-made metallic tin complexes with hydrocarbon substituents with broad applications in food packages, pesticides, wood preservatives, antifouling, and anticorrosion paints. Marine environments near harbors often act as sinks receiving various pollutants from urban runoff, industrial discharge, and atmospheric deposition. This study presented a pioneer analysis of butyltins (BTs) and phenyltins (PhTs) in the Yangtze estuary near one of the largest harbor regions in China. The studied estuary has extensive commercial cargo activities and underdetermined distribution and levels of organotin in sediments and different biota species. Evaluating these factors is essential for determining the risk of exposure and development of future mitigation and possible preventive measures. Seventy-two surface sediments and 13 fish and crab species samples were collected from the Yangtze estuary and its adjacent East China Sea in 2014. BTs and PhTs extracted from samples with ultrasonication treatment were derivatized using sodium tetraethylborate and determined by gas chromatography coupled with mass spectrometer detector. Maximum concentrations of monobutyltin (MBT), dibutyltin (DBT), tributyltin (TBT), diphenyltin, and triphenyltin in sediments were 18.1, 8.9, 6.5, 3.9, and 3.7 ng Sn/g dw (dry weight), respectively. Higher organotin concentrations (e.g., 10-26.4 ng Sn/g dw) were found in the sediments close to the wharfs and fishing ports in the Yangtze estuary, Luchao fishing port, and Yangshan harbor. Organotin concentrations were observed to decrease with increasing distance away from coastlines, highlighting the important roles of human activities in organotin compound(OTC) presence and distribution. The butyltin degradation index indicated a rapid degradation rate of TBT and the existence of input sources for MBT and DBT in the study area. Detection rates and concentrations of organotins in biological samples (i.e., Collichthys lucidus and Carassius auratus) from Luchao port were high, which indicated escalated risks for the environment, ecosystems, and human health.