This study investigated the effects of microwave-assisted (MIC) and ultrasonic-assisted water bath (USWB) pretreatments on the extraction, structural characteristics, physicochemical properties, and thermal stability of Trichosanthes kirilowii Maxim. seed protein isolates (TPI). Results showed that the color difference (ΔE) values of MIC- and USWB-treated TPI were 18.82 and 25.61, respectively. Compared with MIC-TPI, USWB-TPI exhibited a finer and rougher microstructure, a reduced proportion of β-sheet (5.2%), and a looser, more disordered molecular structure. Additionally, USWB-TPI demonstrated higher surface hydrophobicity, superior emulsion stability (19.88 min), enhanced foam stability (70.83%), and improved thermal stability (denaturation temperature (T_d) = 81.05 ℃). Overall, USWB exerted a more pronounced influence on the structural and physicochemical properties of TPI than did MIC. These findings provide valuable guidance for TPI extraction and promote its application in the food industry.