Abstract: Stable strontium isotopes (δ⁸⁸Sr) are sensitive tracers of water–rock interaction, chemical weathering, and dissolved Sr transport, and therefore provide an important complement to conventional radiogenic strontium isotopes (⁸⁷Sr/⁸⁶Sr). Previous studies have mainly focused on riverine, marine, and carbonate systems, whereas investigations of dissolved estuarine waters remain relatively limited. Compared with riverine and marine systems, dissolved waters in estuarine environments are characterized by large salinity gradients, low Sr concentrations, and complex matrix compositions. Consequently, the applicability of existing analytical methods to high-precision stable Sr isotope measurements in such complex estuarine samples, as well as the response of δ88Sr to river–seawater mixing, has not yet been systematically constrained. In this study, dissolved surface, middle, and bottom waters from different salinity zones in the Yangtze River Estuary and adjacent waters were investigated. The δ⁸⁸Sr and ⁸⁷Sr/⁸⁶Sr values were determined using a two-step chemical separation procedure combined with double-spike thermal ionization mass spectrometry (DS-TIMS). Together with salinity, pH, and Sr/Na ratios, these data were used to explore the spatial variation characteristics and controlling mechanisms of dissolved Sr isotopes. The results show that δ⁸⁸Sr generally increases with increasing salinity, whereas ⁸⁷Sr/⁸⁶Sr generally decreases, and both exhibit coordinated variations with the Sr/Na ratio. The isotopic trends are broadly consistent among different water layers, indicating that the analytical procedure is applicable to dissolved estuarine samples with high salinity and low Sr contents. Consistent with previous studies on river–estuary systems in China and elsewhere, the dissolved Sr isotopic composition in the Yangtze River Estuary is mainly controlled by mixing between riverine and seawater endmembers, whereas local deviations in the land–sea interaction zone may be related to local hydrochemical conditions and carbonate-related processes. The combined application of δ⁸⁸Sr and ⁸⁷Sr/⁸⁶Sr is helpful for tracing dissolved Sr transport and river–seawater mixing processes in large estuarine systems.