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Formation characteristics and laws of dislocation and stacking fault during cutting of titanium alloy were investigated by TEM. Several crucial aspects of experiment, such as sample cutting, mechanical reduction of thickness, dimpling, and ion reduction of thickness, were carefully designed and implemented. Further, electron diffraction pattern, diffraction contrast image, and high resolution electron photomicrograph of α phase and β phase were observed and analyzed. Following those analyses, the formation characteristics and laws of dislocation and stacking fault were made clear. Research results show that the edge dislocations exist commonly in the diffraction contrast images and high resolution electron photomicrographs of α phase and β phase. The stacking fault energy is higher in β phase than in α phase. In addition, the extended dislocation is difficult to be seen in β phase, but it is easier to be produced in α phase.