Objective: Previously, we successfully developed a 6-hydrazinonicotinic acid (HYNIC)-modified terazine (Tz) derivative (HYNIC-PEG11-Tz) and labeled with Technetium-99m (99mTc) with a high radiochemical purity. The pretargeted imaging strategy using Atezolizumab-TCO/99mTc-HYNIC-PEG11-Tz is a powerful tool for evaluating Programmed Cell Death Ligand-1 (PD-L1) expression in xenograft mice tumor models. However, the surplus unclicked 99mTc-HYNIC-PEG11-Tz is cleared somewhat sluggishly through the intestines. This is certainly not an ideal situation for imaging for abdominal tumors, especially for colorectal cancer. In order to shift the excretion of the Tz-radioligand to the renal system, we have sought to develop a novel Tz-radioligand by add a polypeptide linker between HYNIC and PEG11. Methods: A polypeptide-modified Tz (HYNIC-Polypeptide-PEG11-Tz) was synthesized and radiolabeled with 99mTc, and the Cetuximab was covalently modified with transcyclooctene (TCO-NHS). The stability of 99mTc-HYNIC-Polypeptide-PEG11-Tz was evaluated in vitro, and its blood pharmacokinetic test was performed in vivo. Determination of the n-octanol/PBS distribution coefficient was performed on 99mTc-HYNIC-PEG11-Tz and 99mTc-HYNIC-Polypeptide-PEG11-Tz to determine the effect of linker modification on their hydrophobicity. In vitro ligation reactivity of 99mTc-HYNIC-Polypeptide-PEG11-Tz towards Cetuximab-TCO was tested. Pretargeted HCT116 cell immunoreactivity binding assay was evaluated. The biodistribution and imaging of 99mTc-HYNIC-Polypeptide-PEG11-Tz was performed to observe the clear pathway of this novel Tz-radioligand. Pretargeted biodistribution of three different accumulation intervals (24 h, 48 h, and 72 h) was performed to determine the optimal pretargeted interval time in nude mice bearing HCT116 tumor xenografts. Comparison of pretargeted (Cetuximab-TCO 48h/99mTc-HYNIC-PEG11-Tz 6h) and (Cetuximab-TCO 48h/ 99mTc-HYNIC-Polypeptide-PEG11-Tz 6h) imaging was performed to show the effect of the two Tz-radioligands with different excretion pathway on tumor imaging. Results: HYNIC-Polypeptide-PEG11-Tz was successfully radiosynthesized with 99mTc, and a radiochemical purity greater than 95% were obtained as confirmed by radio high-performance liquid chromatography (HPLC). 99mTc-HYNIC-Polypeptide-PEG11-Tz showed favorable stability in NS, PBS, and FBS and rapid blood clearance in mice. Liquid chromatograph-mass spectrometer (LC-MS) indicated the presence of an average 8.1 TCO moieties per Cetuximab. Size exclusion HPLC revealed almost complete reaction between Cetuximab-TCO and 99mTc-HYNIC-Polypeptide-PEG11-Tz in vitro, with the 8:1 Tz-to-mAb reaction providing a conversion yield of 87.83±3.27%. Pretargeted cell immunoreactivity binding assay showed high affinity to HCT116 cells. The biodistribution and imaging of 99mTc-HYNIC-Polypeptide-PEG11-Tz demonstrated that the Tz-radioligand was cleared through kidneys. After allowing 24h, 48h and 72h for accumulation of Cetuximab-TCO in HCT116 tumor, pretargeted biodistribution revealed an uptake of the radiotracer in the tumor with tumor-to-blood ratio of 0.83, 1.40, and 1.15, respectively. Both pretargeted (Cetuximab-TCO 48h/99mTc-HYNIC-PEG11-Tz 6h) and (Cetuximab-TCO 48h/ 99mTc-HYNIC-Polypeptide-PEG11-Tz 6h) imaging delineated the HCT116 tumor clearly. However, pretargeted imaging strategy using Cetuximab-TCO 48h/99mTc-HYNIC-Polypeptide-PEG11-Tz 6h) could be used for diagnosing colorectal cancer since the surplus unclicked 99mTc-HYNIC-Polypeptide-PEG11-Tz is cleared through urinary system and produces low abdominal uptake background. Conclusion: We developed a novel pretargeted imaging strategy (Cetuximab-TCO/99mTc-HYNIC-Polypeptide-PEG11-Tz) for imaging colorectal cancer since the surplus unclicked 99mTc-HYNIC-Polypeptide-PEG11-Tz produces low abdominal uptake background, which broadens the application scope of pretargeted imaging strategy.