Abstract: Gel retardation, also named electrophoretic mobility shift assay (EMSA) , is a useful tool for identifying protein-DNA interactions. Typically, ³²P-labeled DNA probes used in EMSA are sensitive. However, it relies on the handling of hazardous radioisotopes, and is not easily quantified. Recently, some successful cases have been reported using non-radio labelled probes instead of radiolabelled probes in EMSA. The method is rapid, convenient, and safe, but it depends on a very expensive kit. In this study, we offered a new method performing EMSA by modifying DIG High Prime DNA Labeling and Detection Starter Kit II (Rohe). Firstly, the prepared labeled probe was introduced the EcoRI stick in the end of probe for 3`-end labeling , and then was performed the probe labeling and detecting the signals of EMSA with the relatively cheap DIG High Prime DNA Labeling and Detection Starter Kit II （Rohe）. By adjusting the experiment parameters, the successful result was obtained. The present study provides a successful example and method for modifying DIG High Prime DNA Labeling and Detection Starter Kit II.