The impacts of industrial phosphorylation on the structural changes, microstructure, functional, and rheological features of soybean protein isolate (SPI) were spotlighted. The findings implied that the spatial structure and functional features of the SPI changed significantly after treatment with the two phosphates. Sodium hexametaphosphate (SHMP) promoted aggregation of SPI with a larger particle size; sodium tripolyphosphate (STP) modified SPI with smaller particle size. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) results showed insignificant alterations in the structure of SPI subunits. Fourier transform infrared (FTIR) and endogenous fluorescence noted a decline in α-helix quantity, an amplification in β-fold quantity, and an increase in protein stretching and disorder, indicating that phosphorylation treatment fluctuated the spatial structure of the SPI. Functional characterization studies showed that the solubility and emulsion properties of the SPI increased to varying degrees after phosphorylation, with a maximum solubility of 94.64% for SHMP-SPI and 97.09% for STP-SPI. Emulsifying activity index (EAI) and emulsifying steadiness index (ESI) results for STP-SPI were better than those for SHMP-SPI. Rheological results showed that the modulus of G' and G″ increased and the emulsion exhibited significant elastic behavior. This affords a theoretical core for expanding the industrial production applications of soybean isolates in the food and various industries.