Radiolabeled peptide could be a useful tool for the diagnosis of non‐small‐cell lung cancer (NSCLC). In this study, HYNIC‐(Ser)₃‐J18 peptide was labeled with ^99mTc using EDDA/tricine as coligands. The in vitro and in vivo studies of this radiolabeled peptide were performed for cellular‐specific binding and tumor targeting in A‐549 cells and tumor‐bearing mice, respectively. The high radiochemical purity was obtained and this radiolabeled peptide exhibited high stability in buffer and serum. The radiolabeled peptide showed high affinity for the A‐549 cells with a dissociation constant value (K_D) of 4.4 ± 0.8 nm. The tumor–muscles ratios were 2.7 and 4.4 at 1 and 2 hr after injection of ^99mTc‐(EDDA/tricine)‐HYNIC‐(Ser)₃‐J18 in tumor‐bearing mice. The tumor uptake was decreased after preinjection with non‐labeled peptide for this radiolabeled peptide in blocking experiment. The results of this study showed the ^99mTc‐(EDDA/tricine)‐(Ser)₃‐HYNIC‐J18 peptide might be a promising radiolabeled peptide for NSCLC targeting.