Abstract: Hypoxic cells within a tumor is associated with the radiotherapy and chemotherapy resistance. The development of noninvasive methods for detecting hypoxia is need. ⁹⁹Tc^m-BnAO is a nonnitroimidazole imaging agent for hypoxia，and has been evaluated both in vitro and in vivo as a noninvasive marker. In order to improve its ability for imaging, the ligand BnAO was modified with ethylene glycol monoethyl ether (EGME) on both sides. BnAO and BnAOEGME were labeled with ⁹⁹Tc^m. ⁹⁹Tc^m-BnAO and ⁹⁹Tc^m-BnAO-EGME were found to be electrically neutral at physiological condition by paper electrophoresis. ⁹⁹Tc^m-BnAO-EGME showed higher hydrophilicity in octanol/water partition coefficient experiment and better stability in vitro. The biodistribution in mice bearing S180 tumor demonstrated that⁹⁹Tc^m-BnAO and ⁹⁹Tc^m-BnAO-EGME could accumulate in tumor with time going. However, it was found that ⁹⁹Tc^m-BnAO-EGME showed a little disadvantage in the ratio tumor to blood and the ratio of tumor to muscle compared with ⁹⁹Tc^m-BnAO.