With an attempt to economically and efficiently improve the water resistance of defatted soybean flour (DSF)-based wood adhesives, DSF was subjected to thermal treatment at various temperatures (65, 80, 95, 110, and 125 °C) for 30 minutes. The effects of thermal treatment temperature onto the chemical structure, crystalline degree, water-insoluble content, and acetaldehyde value of the thermally-treated DSF (T-DSF) was investigated. The thermal stabilities and bonding properties of soybean adhesives prepared from T-DSF and crosslinker epichlorohydrin-modified polyamide (EMPA) were also investigated. Test results indicated that both the water insoluble content and acetaldehyde value of T-DSF increased after thermal treatment, reaching the highest values of 27.28% and 26.81 mmol/g, respectively. All plywood bonded with the T-DSF-based adhesive withstood a 28 h boiling–dry–boiling accelerated aging treatment, while plywood bonded with the DSF-based adhesive delaminated after 4 h of water boing, demonstrating the significantly improved water resistance of the T-DSF-based adhesives. Related analyses also confirmed that this improvement was due to: (i) the formation of insoluble crosslinked structures of T-DSF resulting from protein–protein self-crosslinking reactions and the protein–carbohydrate Maillard reaction, and (ii) increased crosslinking efficiency between T-DSF and crosslinker EMPA owing to more T-DSF reactive groups being released after thermal treatment.