Human epidermal growth factor receptor type 2 (HER2) is abundant in a wide variety of tumors and associated with the poor prognosis. Radiolabeled affibodies are potential candidates for detecting HER2-positive lesions. However, laborious multiple-step synthetic procedure and high abdomen background may hinder the widespread use. Herein, cysteinylated ZHER modified with a new hydrophilic linker (denoted as MZHER) was designed and labeled using FAl-NOTA strategies. The biologic efficacy of the novel tracer and its feasibilities for in vivo monitoring HER2 levels were also investigated in xenograft models with different HER2 expressions. MZHER was conjugated with MAL-NOTA under standard reaction conditions. The affibody molecule was then radiolabeled with FAl complex. The binding specificity of the tracer, FAl-NOTA-MAL-MZHER, with HER2 was primarily characterized via in vitro studies. MicroPET imaging were performed in nude mice bearing tumors (SKOV-3, JIMT-1 and MCF-7) after injection. The HER2 levels of xenografts were determined using Western blotting analysis. FAl-NOTA-MAL-MZHER can be efficiently produced within 30 min with a non-decaycorrected yield of about 10% and a radiochemical purity of more than 95%. In vitro experiments revealed that the modified affibody retained the specific affinity to HER2. PET imaging showed that SKOV-3 and JIMT-1 xenografts were clearly visualized with excellent contrast and low abdomen backgrounds. On the contrary, the signals of MCF-7 tumor were difficult to visualize. The ROI values ranged from16.54±2.69% ID/g for SKOV-3 to 8.42±1.20 %ID/g for JIMT-1 tumors at 1h postinjection respectively. Poor uptake was observed from MCF-7 tumors with 1.71±0.34% ID/g at the same time point. Besides, a significant linear correlation between % ID/g values and relative HER2 expression levels was also found. FAl-NOTA-MAL-MZHER is a promising tracer for in vivo detecting HER2 status with the advantages of facile synthesis and favorable pharmacokinetics. It may be useful in differential diagnosis, molecularly targeted therapy and prognosis of the cancers.