DNA conjugated gold nanorods (AuNRs) are widely applied far nanostructure assembly, gene therapy, biosensing and drug delivery. However, it is still a great challenge to attach thiolated DNA on AuNRs, because the positively charged AuNRs readily aggregate in the presence of negatively charged DNA. This article reports an mPEG-SH/Tween 20-assisted method to load thiolated DNA on AuNRs in 1 h. Tween 20 and mPEG-SH are used to synergistically displace CTAB on the surface of AuNRs by repeated centrifugation and resuspension, and thiolated DNA are attached to AuNRs in the presence of 1 M NaCl, 100 mM MgCl2, or 100 mM citrate. AuNRs with different sizes and aspect ratios can be functionalized with DNA by this method. The number of DNA loaded on each AuNR can be easily controlled by the concentrations of mPEG-SH and Tween 20 or the ratio between DNA and AuNR. Functionalized AuNRs were used for nanoparticle assembly and cancer cell imaging to confirm that DNA anchored on the surface of AuNRs retains its hybridization and molecular recognition capability. The new method is easy, rapid, and robust for the preparation of DNA functionalized AuNRs for a variety of applications such as cancer therapy, drug delivery, self-assembly, and imaging.