The thermal aggregation behavior of soy protein isolate (SPI) has a significant impact on the functional and nutritional properties of soy protein based-food. In this study, the capability of asymmetrical flow field-flow fractionation (AF4) combined with sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) for monitoring the thermal aggregation behavior of SPIs in situ was evaluated. The effects of heating temperature and time, protein concentration, ionic strength and type of salt on the thermal aggregation behavior of SPIs were investigated. Two types of SPIs subunits aggregates with different sizes and compositions in this work were observed, which mainly owes to the utilization of open channel in AF4 as well as SDS-PAGE. Furthermore, the results suggested that larger SPIs aggregates can dissociate, rearrange, and aggregate to form the smaller ones at a long heating time. Zeta potential and turbidity results revealed that higher ionic strength promotes the formation of larger SPIs aggregates. AF4 combined with SDS-PAGE was proved to be a powerful tool for studying the thermal aggregation behavior of SPIs.