The Indonesian Throughflow regulates the heat and freshwater budgets between the western Pacific and Indian Oceans, playing an important role in low latitude and global climate change. Modern oceanographic studies show that the transport of the Indonesian Throughflow mainly happens in the thermocline, but few studies explored its evolution history and driving mechanism. In this study, sediments from Core SO18526, which was drilled at the Makassar Strait within the main inflow passage of the Indonesian Throughflow, were used as the study material. We reconstructed the evolution history of the thermocline seawater temperature and salinity and depth of thermocline at the coring site of Core SO18526 during the past 25 ka based on shell δ18O and Mg/Ca of planktonic foraminifera thermocline-dwelling species Pulleniatina obliquiloculata. By integrating new records with published paleo-oceanographic and climatological records, we found that variations in the thermocline seawater temperature and salinity and depth of thermocline at the coring site of Core SO18526 are regulated by the mean state of El Niño-Southern Oscillation-like conditions on glacial-interglacial timescale. Moreover, decreased temperature and salinity of the thermocline seawater and shoaled thermocline at the coring site of Core SO18526 could be attributed to the increased regional rainfall and the flooding of the Sunda shelf since 8.5 ka.