CXCR4 plays an important role in a number of immunological-based diseases and cancers. The development of radiotracers targeting CXCR4 can provide a valuable tool for the diagnosis and monitoring of conditions involving deregulation of the receptor. The incorporation of multiple radionuclides into small molecules, peptides, and CXCR4 ligands have been reported and characterized. In this study, a 99mTc labeled AMD3465 with high yields and radiochemical purity was prepared. In vitro and in vivo study of [99mTc]TcAMD3465 showed high stability and binding affinity, with a hydrophilic log P value of − 2.67. Biodistribution study was investigated using tissue and organ samples with a well-type Na(I) detector, high radioactivity accumulation was observed in liver, spleen and bone, consistent with high CXCR4 expression in these organs. SPECT imaging was performed in H460 xenograft mouse model, [99mTc]TcAMD3465 showed comparable tumor uptake and a significantly high tumor-to-background ratio, suggesting potential utility of [99mTc]TcAMD3465 as a CXCR4 receptor imaging agent.