Local recurrence is a common cause of treatment failure for patients with solid tumors. Tumor-specific intraoperative fluorescence imaging may improve staging and debulking efforts in cytoreductive surgery and, thereby improve prognosis. Here, we report in vivo assembly of the second near-infrared window (NIR-II) emitting downconversion nanoparticles (DCNPs) modified with DNA and targeting peptides to improve the image-guided surgery for metastatic ovarian cancer. The NIR-II imaging quality with DCNPs is superior to that of clinically approved ICG with good photostability and deep tissue penetration (8 mm). Stable tumor retention period experienced 6 h by in vivo assembly of nanoprobes can be used for precise tumor resection. Superior tumor-to-normal tissue ratio is successfully achieved to facilitate the abdominal ovarian metastases surgical delineation. Metastases with ≤1 mm can be completely excised under NIR-II bioimaging guidance. This novel technology provides a general new basis for the future design of nanomaterials for medical applications.