Osteosarcoma is an aggressive tumor of mesenchymal origin that is more likely to spread to the lung than others, with a major impact on patients' prognosis. The optimal imaging method that can reliably detect or exclude pulmonary metastases from osteosarcoma is still scarce. Herein, two homologous types of fluorescent probes CH1055-PEG-PT and CH1055-PEG-Affibody, which show highly promising results for targeting imaging of osteosarcoma and its lung metastasis, respectively, are designed and synthesized. It is found that the NIR-II imaging quality of CH1055-PEG-PT is far superior to that of computed tomography for the early in vivo 143B tumor imaging, and this probe-guided surgery for accurate resection of 143B tumor is further performed. The high-resolution visualization of primary and micrometastatic lung lesions of osteosarcoma by using CH1055-PEG-Affibody is also demonstrated. Therefore, the attractive imaging properties of CH1055-PEG-PT and CH1055-PEG-Affibody, including high levels of uptakes, and high spatial and temporal resolution, open up opportunities for molecular imaging and clinical translation of osteosarcoma and its lung metastasis in the unique second near-infrared window.