Positron emission tomography (PET) is a common imaging technique and can provide accurate information about the size, shape, and location of tumors. Recent evidence has shown that G-quadruplex structures (G4s) are identified in human oncogenes, and these special structures are recognized as diagnostic cancer markers and drug targets for anticancer therapies. Although a number of techniques for in vivo imaging of G4s have been developed, achieving sufficient sensitivity and selectivity in vivo remains challenging. Herein, we have engineered and developed a radiolabeled peptide probe [18F]AlF-NOTA-RHAU18 targeting mitochondrial DNA G4s for in vivo PET imaging. The results of the study indicate that this probe is able to visualize and detect solid tumors in living homozygous mice. In addition, the distribution of the probe in cancer cells was investigated using FITC-RHAU18. This work may offer new insights into the development of cancer diagnostic tools by targeting in vivo G4s.