Abstract: ¹²⁵I-Atezolizumab was synthesized with Iodogen method for monitoring the expression level of PD-L1 in tumors. The influence of reaction time on ¹²⁵I-labeling efficiency and the stability of ¹²⁵I-Atezolizumab in PBS and fetal bovine serum were studied. The pharmacokinetics of ¹²⁵I-Atezolizumab was evaluated in rats. Binding specificity to PD-L1 was determined using cellular uptake experiment of breast cancer cell line MDA-MB-231 in vitro and SPECT imaging of mice bearing MDA-MB-231 xenografts in vivo. The results indicated that the labeling yield of ¹²⁵I-Atezolizumab was over 98% after 5 minutes reaction at room temperature. The radiochemical purity was more than 90% after 48 h incubation in PBS and fetal bovine serum. The elimination half-life of ¹²⁵I-Atezolizumab in rats was (12.1±1.9) h. The immunological activity of ¹²⁵I-Atezolizumab was well maintained (IF=52%) and high affinity was demonstrated in breast cancer cell line. SPECT/CT imaging of MDA-MB-231 tumor-bearing mice showed high radioactivity uptake in the tumor. Tumor uptake of ¹²⁵I-Atezolizumab was blocked in the presence of Atezolizumab, confirming target specificity. The preliminary results suggested that ¹²⁵I-Atezolizumab exhibited specificity for PD-L1 imaging using a simple and efficient labeling method, and demonstrated relatively good in vitro stability. It is potential to monitor the expression levels of PD-L1 in tumors by ¹²⁵I-Atezolizumab SPECT imaging and is worthy of further research.