This work reports, a novel method of peanut tofu preparation by combining transglutaminase (TG) and high-temperature pressure cooking (HTPC, 115 °C, 0.17 MPa) treatment, and its gel formation was studied. Texture and Scanning electron microscope (SEM) results showed TG addition (0.75 mg/g) significantly improved the tofu texture (p < 0.05), with a denser network composed of relatively even pores, whereas the excessive addition (1.25 mg/g) resulted in a softer tofu. At all concentrations (0.25–1.25 mg/g), TG treatment induced the crosslinking of protein molecules as proved by SDS-PAGE, particle size and apparent viscosity analyses. DSC measurement indicated that the subsequent HTPC treatment was able to denature peanut proteins completely. Moreover, by analyzing the changes in surface hydrophobicity (H0) and total free sulphydryl content (TFS) during processing, we found that hydrophobic interactions and disulfide bonds were the major forces involved in the formation of peanut tofu gel. This study suggests this novel method could be applied to make peanut tofu and provides a basis for understanding its gel formation process.