Chemokine receptor (CXCR-4) is overexpressed in many types of tumors and promotes cancer metastasis. The expression level of CXCR4 is the sign of the metastatic potential of the tumor and determines organ-specific metastasis. This chemokine receptor is considered a promising target for cancer treatment and molecular imaging. In this study, a cyclo-peptide as CXCR4 antagonist was successfully synthesized applying a standard Fmoc solid-phase strategy and radio-labeled with Sodium Pertechnetate via HYNIC chelator and EDDA/tricine co-ligands. The ligand was manually synthesized in high yield (RP-HPLC:>95%) and its chemical structure was confirmed using Mass spectroscopy ([M+H]⁺=1154.60). RTLC and HPLC analysis of radiolabeled peptides indicated more than 95% radiochemical purity. Incubated [^99mTc]-HYNIC-peptide in human serum for 24 hr at 37 °C indicated high stability of more than 95%. Scatchard plot of radio-ligand in B16-F10 cells showed favorable affinity (Kd=50 μmol and Bmax=0.0182 (pmol/mg protein) for the cell membrane. Biodistribution evaluation showed 1.84±0.13 % ID and 3.49±0.27 %ID in tumor tissue at 30 min post-injection with and without plerixafor as receptor blocker, respectively which shows high affinity and selectivity of the radioligand to CXCR4 receptors. Planar imaging indicated high uptake of radioligand in implanted tumor tissue in mice. The findings reveal that the introduced radiolabelled peptide might be a potential SPECT imaging agent for high proliferated CXCR4 tumors to accurate cancer prognosis.