The objective of this study was to investigate the effects of ultrasound on the structure and function of human tumor necrosis factor-alpha (TNF-alpha) and to study whether TNF-alpha underwent a denaturation process and the molecular structure was damaged when it was irradiated by ultrasound. The samples of TNF-alpha were dissolved in aqueous solution and filled into polystyrene tubes. High intensity ultrasound processor (20 kHz frequency, burst mode, 0.5 duty factor, 100-500 W total electrical power, 0-20 min total treatment time) was used during the treatment. The biologic activity of TNF-alpha was determined by its toxic activity towards TNF-alpha sensitive cell line L929 in the presence of actinomycin D. The methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) were used to detect the integrity of TNF-alpha molecule after it was irradiated by ultrasound. The results showed TNF-alpha could keep its biological activity, instead of undergoing a denaturation process, when it is irradiated by ultrasound in the aqueous solution; at the same time, the aggregates of TNF-alpha formed by the recombinant DNA E. coli could be dissociated through the molecular vibration induced by ultrasound energy. The biologic activity of TNF-alpha was not reduced, but small quantities of TNF-alpha molecular structure were damaged during the process of sonication. These features of TNF-alpha molecule irradiated by ultrasound probably gave TNF-alpha the advantage in being used in the drug microencapsulation and provided a new drugs formulation for tumor therapy.