The Southern blot analysis based on isotope labeling technique has some shortages such as facility limitation, environmental pollution, and poor stability. Therefore, establishing a safe, stable and efficient Southern hybridization protocol is very important to detect the integration of exogenous genes in transgenic wheat. In this study, by using the DNAs from wild-type wheat and transgenic wheat as material, the DIG-labeled Southern blot analysis was improved through modifying several key steps, including probe preparation and purification, usage of DNA samples amount, digestion system, vacuum transfer conditions, stringent hybridization conditions, and immunoassay detect. The results showed that the purifications of DNA template and probe- labeled could significantly improve the efficiency of the probe labeling when random prime labeling method was used. Desirable digestion result could be achieved when 10μg DNA samples with high quality was enzymed in 80μl reaction system for 8～12 hours. In the vacuum transfer step, alkaline liquid was better than neutral liquid to obtain clean transmembrane effect. Reagent purity, the temperature and the rotation speed in hybridization inside the hybridization oven impacted on the hybridization results greatly. Application of chemiluminescence detection system combining with improved coating CSPD method was not only easier to operate, but also cleaner in background comparing X-ray imaging optimized. The improved digoxigenin-labeled Southern blot technique showed good sensitivity and high ratio of signal to noise for wheat DNA analysis, can be used stably and widely in ordinary laboratory, overcoming restrictions of isotope labeling to experimental conditions, equipments, and physical status of researcher.