Angiotensin-converting enzyme 2 (ACE2) is not only a key to the renin-angiotensin-aldosterone system and related diseases, but also the main entry point on cell surfaces for certain coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2. By analyzing the different key binding sites from the receptor-binding domain (RBD) of SARS-CoV and SARS-CoV-2, nine new ACE2-targeting peptides (A1 to A9) were designed, synthesized and connected with a chelator, 1,4,7-triazacyclononane-N,N',N''-triacetic acid (NOTA). NOTA-A1, NOTA-A2, NOTA-A4, NOTA-A5, and NOTA-A8 were successfully labeled with [68Ga]Ga3+ and were used for biological evaluation. [68Ga]Ga-NOTA-A2, [68Ga]Ga-NOTA-A5, and [68Ga]Ga-NOTA-A8 showed specific binding to ACE2 via cell assays, and their binding sites and binding capacity were calculated by molecular docking and molecular dynamics simulations. In tumor-bearing mice, A549 tumors were visualized 60 min postinjection of [68Ga]Ga-NOTA-A2, [68Ga]Ga-NOTA-A5, or [68Ga]Ga-NOTA-A8. These peptides also accumulated in the organs with high-level ACE2 expression, confirmed by immunohistochemical stain. Among them, [68Ga]Ga-NOTA-A5 exhibited the highest tumor uptake and tumor/background ratio, and it successfully tracked the increased ACE2 levels in mice tissues after excessive Losartan treatment. In a first-in-human study, the distribution of [68Ga]Ga-NOTA-A5 was evaluated with positron emission tomography/computed tomography (PET/CT) in three participants without adverse events. 68Ga-labeled peptides originated from the coronavirus RBD, with [68Ga]Ga-NOTA-A5 as a typical representative, seem to be safe and effective for the evaluation of ACE2 expression in vivo with PET/CT, facilitating further mechanism investigation and clinical evaluation of ACE2-related diseases.