The elevated expression of urokinase-type plasminogen activator receptor (uPAR) is associated with the poor prognosis of pancreatic cancer patients. Thus, uPAR is a promising candidate as a molecular target for the non-invasive imaging of pancreatic cancer. The present study aimed to develop a technetium-99m (99mTc)-labeled uPAR-binding peptide for non-invasive single-photon emission computed tomography (SPECT) assessment of uPAR expression in pancreatic cancer xenograft models. A linear high-affinity uPAR peptide antagonist, Hynic-PEG-AE105, was labeled with 99mTc. Human uPAR-positive pancreatic cancer BxPC-3 cells were inoculated into nude mice. SPECT was performed in the pancreatic cancer xenograft mice models. The results showed that the rate of the 99mTc labeling of Hynic-PEG-AE105 was 97.72±1.73%. The tumor uptake of 99mTc-Hynic-PEG-AE105 was higher than the control inactive peptide 99mTc-Hynic-PEG-AE105mut at 4 h (3.37±0.11 vs. 1.36±0.18; P<0.001) and 6 h (3.64±0.25 vs. 1.28±0.20; P<0.001) (n=10). Moreover, a significant correlation was observed between the tumor uptake of 99mTc-Hynic-PEG-AE105 and uPAR expression (r=0.791, P=0.006). In conclusion, in the present study, a peptide-based SPECT tracer, 99mTc-Hynic-PEG-AE105, with a high purity and specific radioactivity was synthesized. 99mTc-Hynic-PEG-AE105 is a promising agent for the non-invasive determination of uPAR expression in pancreatic cancer.