To regulate the degradation of transgenic DNA and lay theoretical foundations for the rational utilization of genetically modified (GM) products, variations in copy numbers and structural characteristics of DNA from GM soybean event GTS 40-3-2 during soybean protein concentrate (SPC) preparation were evaluated. Results showed that defatting and the first ethanol extraction were key procedures inducing DNA degradation. After these two procedures, copy numbers of thelectinandcp4epspstargets decreased by more than 4 × 108, occupying 36.88–49.30% of the total copy numbers from raw soybean. Atomic force microscopy images visually revealed the degradation of DNA that thinned and shortened during SPC preparation. Circular dichroism spectra suggested a lower helicity of DNA from defatted soybean kernel flour and a conformation transition of DNA from B-type to A-type after ethanol extraction. The fluorescence intensity of DNA decreased during SPC preparation, verifying the DNA damage along this preparation chain.