This study aimed to explore the effects of microwave and anionic surfactant sodium dodecyl sulfate (SDS) modification on the structure of soy protein isolate and its film-forming properties. Microwave and SDS were used to treat soy protein isolate (SPI) film-forming solution and films were prepared by casting method. The results demonstrated that microwave treatment led to partial unfolding in the structure of SPI, exposing more reactive groups and the hydrophobic groups hidden inside SPI. This change promoted formation of hydrogen bonds among the side chains of SPI as well as hydrophobic bonds and electrostatic bonds between SPI and SDS, forming cross-linked spatial network structure. Meanwhile, hydrogen bonds formed between glycerol and SPI stabilized this network structure. SPI-based films treated by microwave exhibited higher mechanical properties and better surface hydrophobicity than the untreated ones. Film with microwave treatment at the power of 400 W displayed the highest tensile strength and elongation at break, which were increased by 61.91% and 453.75%, respectively. In addition, these films also showed relatively high surface hydrophobicity. Microwave and SDS modified SPI-based films showed improved mechanical properties and better surface hydrophobicity.