The early diagnosis of non-small cell lung cancer (NSCLC) is important for increasing survival rate and improving quality life of patients. The aim of this study was to investigate ^99m Tc-(tricine)-HYNIC-(Ser)₃-J18 for targeting and imaging of NSCLC in A-549 xenografted nude mice. The (Ser)₃-J18 peptide was conjugated with HYNIC and labeled with ^99m Tc using tricine as a co-ligand. The radiolabeled peptide was evaluated for its radiochemical purity, stability, receptor binding and internalization in vitro. The future experiments were performed for tumor targeting and imaging in A-549 tumor-bearing mice. ^99m Tc-(tricine)-HYNIC-(Ser)₃-J18 was obtained at high labeling efficiency at room temperature and favorable stability in saline and human plasma. At the cellular level, the radiolabeled peptide specifically bond to A-549 cells with a K_D 4.1 ± 1.3 nM. Biodistribution study revealed tumor to blood and tumor to muscle ratios were about 3.12 and 5.63 respectively after 2 h injection of radiolabeled peptide. These ratios were significantly decreased by co-injection of excess non-labeled peptide in mice. This radiolabeled peptide selectively targeted to NSCLC tumor and exhibited a high target uptake combined with acceptable low background activity for tumor imaging in mice. The results of this study and its comparison with another study showed that ^99m Tc-(tricine)-HYNIC-(Ser)₃-J18 is better than previously reported radiolabeled peptide as ^99m Tc-(EDDA/tricine)-HYNIC-(Ser)₃-J18 for NSCLC targeting and imaging.