Cetuximab is commonly used to treat patients with advanced gastric cancer and the overall level of tumor epidermal growth factor receptor (EGFR) expression critically impacts the efficacy of cetuximab. This study was performed to select the most clinically relevant probes targeting EGFR via comparative research. Cetuximab was fragmented by enzymatic cleavage to produce Cet-F(ab')₂ and Cet-Fab, which were then conjugated with CY3 NHS ester to obtain CY3-Cetuximab, CY3-Cet-F(ab')₂, and CY3-Cet-Fab for cell fluorescence experiments to verify their affinity. Three ¹²⁴I-labeled molecular probes, namely ¹²⁴I-Cetuximab, ¹²⁴I-Cet-F(ab')₂, and ¹²⁴I-Cet-Fab, were prepared using the Iodogen method. Micro-PET/CT imaging was performed 1 h, 3 h, 6 h, and 12 h after probe injection for comparative studies. Further, biological distribution experiments were conducted to explore the in vivo distributions of the three probes at different time points. Independent sample t-tests were used for intergroup comparisons. Fragmentation did not significantly decrease cetuximab affinity. The respective molecular weights of Cetuximab, Cet-F(ab')₂, and Cet-Fab were approximately 150 kDa, 100 kDa, and 50 kDa, respectively. After ¹²⁴I labeling and purification, the radiochemical purity of ¹²⁴I-Cetuximab, ¹²⁴I-Cet-F(ab')₂, and ¹²⁴I-Cet-Fab exceeded 94%, and the specific activity was approximately 2×10⁴ MBq/μmol. Stability tests in saline and fetal bovine serum (FBS) showed that radiochemical purity remained above 90% after 24 h of incubation. Micro-PET/CT imaging demonstrated significant accumulation of ¹²⁴I-Cet-Fab at the tumor site 3 h after intravenous injection, whereas ¹²⁴I-Cet-F(ab')₂ exhibited pronounced accumulation at the tumor site 12 h after injection. However, no evident distribution of the imaging agent was observed at the tumor site 12 h after intravenous injection of ¹²⁴I-Cetuximab. The biological distribution results are consistent with the micro-PET/CT findings. Among the three positron probes, ¹²⁴I-Cet-Fab exhibited the fastest metabolism, earliest tumor imaging, and the lowest cumulative radiation dose, indicating its potential for clinical translation.