The chlorambucil l-histidine conjugate was synthesized and radiolabeled with [99mTc(CO)3]+ core to form the 99mTc(CO)3(His–CB) complex. The radiochemical purity of the complex was over 90%. It had good hydrophilicity and was stable at room temperature. The high initial tumor uptake with certain retention, fast clearance from background, good tumor/non-tumor ratios and satisfactory scintigraphic images highlighted the potential of 99mTc(CO)3(His–CB) as a tumor imaging agent.