A first-in-human study evaluated the safety, biodistribution, and dosimetry of site-specifically labeled 89Zr-pertuzumab (89Zr-ss-pertuzumab) in patients with HER2-positive breast cancer. Six patients with HER2-positive metastatic breast cancer were enrolled in a prospective clinical trial. Pertuzumab was site-specifically modified with desferrioxamine (DFO) using a novel chemoenzymatic strategy and then labeled with 89Zr. Patients received 74 MBq of 89Zr-ss-pertuzumab intravenously and underwent PET/CT imaging at 1, 3–4, and 5–8 days after injection. The study demonstrated that 89Zr-ss-pertuzumab PET/CT was safe, with no toxicities observed. The optimal imaging time point was 5–8 days after administration, and the radiotracer showed high uptake in HER2-positive lesions. Dosimetry estimates indicated that the organs receiving the highest doses were the kidney, liver, and heart wall. The average effective dose for 89Zr-ss-pertuzumab was 0.54 ± 0.03 mSv/MBq, comparable to 89Zr-DFO-pertuzumab and 89Zr-DFO-trastuzumab. 89Zr-ss-pertuzumab demonstrated improved lesion detection and higher tracer avidity compared to 89Zr-DFO-pertuzumab. The study concluded that 89Zr-ss-pertuzumab PET/CT is a promising tool for assessing HER2 status in patients with HER2-positive breast cancer, potentially guiding biopsy and treatment decisions. The results suggest that 89Zr-ss-pertuzumab may detect more lesions than 89Zr-DFO-pertuzumab, and its potential clinical applications include real-time evaluation of HER2 status to guide biopsy and treatment decisions.A first-in-human study evaluated the safety, biodistribution, and dosimetry of site-specifically labeled 89Zr-pertuzumab (89Zr-ss-pertuzumab) in patients with HER2-positive breast cancer. Six patients with HER2-positive metastatic breast cancer were enrolled in a prospective clinical trial. Pertuzumab was site-specifically modified with desferrioxamine (DFO) using a novel chemoenzymatic strategy and then labeled with 89Zr. Patients received 74 MBq of 89Zr-ss-pertuzumab intravenously and underwent PET/CT imaging at 1, 3–4, and 5–8 days after injection. The study demonstrated that 89Zr-ss-pertuzumab PET/CT was safe, with no toxicities observed. The optimal imaging time point was 5–8 days after administration, and the radiotracer showed high uptake in HER2-positive lesions. Dosimetry estimates indicated that the organs receiving the highest doses were the kidney, liver, and heart wall. The average effective dose for 89Zr-ss-pertuzumab was 0.54 ± 0.03 mSv/MBq, comparable to 89Zr-DFO-pertuzumab and 89Zr-DFO-trastuzumab. 89Zr-ss-pertuzumab demonstrated improved lesion detection and higher tracer avidity compared to 89Zr-DFO-pertuzumab. The study concluded that 89Zr-ss-pertuzumab PET/CT is a promising tool for assessing HER2 status in patients with HER2-positive breast cancer, potentially guiding biopsy and treatment decisions. The results suggest that 89Zr-ss-pertuzumab may detect more lesions than 89Zr-DFO-pertuzumab, and its potential clinical applications include real-time evaluation of HER2 status to guide biopsy and treatment decisions.