Abstract: Phosphatidylethanolamine (PE) is externalized to the cell surface during the early stages of apoptosis. Duramycin is a peptide that can bind to PE with high selectivity and specificity. Radionuclide labeled Duramycin enables in vivo imaging to monitor apoptosis at an early stage. In this study, the imaging agent ^99mTc-HYNIC-Duramycin was obtained by one-step reaction between ^99mTc and the precursor HYNIC-Duramycin. The radiolabeling process takes approximately 20 min, and the radiochemical purity is above 95%. ^99mTc-HYNIC-Duramycin is water-soluble, with a LogP value of −1.73±0.06 (n=3), and the radiochemical purity remains above 95% in saline for up to 6 h, indicating excellent stability in vitro. Biodistribution studies showed that ^99mTc-HYNIC-Duramycin is primarily metabolized by the liver, with higher radioactive accumulation observed in the kidneys and spleen, and lower uptake in blood and muscle. Compared with the control group and the blank group, ^99mTc-HYNIC-Duramycin has significantly higher uptake in NCI-H460 tumor after cisplatin treatment by SPECT/CT imaging. These results demonstrate the good specificity of the imaging agent and its ability to monitor chemotherapy-induced apoptosis in tumors at an early stage. In conclusion, ^99mTc-HYNIC-Duramycin is a promising SPECT/CT radiotracer for early treatment evaluation following chemotherapy.