Although the potential value of phenotypic/functional knockout technology with intrabody/kine in prevention and cure of some serious diseases, such as AIDS and cancer, is being regarded, there are still several technical difficulties. One of the the most critical problems is how to directly deliver the intrabody/kine proteins into endoplasm reticulum (ER). In this study, a novel recombinant protein, TAT-GFP-KDEL, was designed and constructed. In this recombinant protein, HIV-derived TAT (47-57) and an ER retention four-peptide sequence KDEL were fused at the N-terminal and C-terminal of GFP respectively. The results showed that TAT-GFP-KDEL had been successfully expressed in bacteria BL21 and its purity reached to 95%. Moreover, we observed that this recombinant protein was able to efficiently transduce into MOLT-4 cells and accurately locate at ER. This study may provide an available strategy to promote the transmembrane delivery and ER localization of protein-based intrabody/kine.