Efficacious cancer treatment largely relies on accurate imaging diagnosis and imaging-guided surgery, which can be achieved by combining different mode imaging probes on one single nanoplatform. Herein, a novel radiolabeled NIR-II nanoprobe (125I-MT NP) was developed to enable versatile single-photon emission computed tomography (SPECT) and second near-infrared (NIR-II) fluorescence dual-modal imaging against breast cancer. 125I-MT was precipitated with an amphiphilic triblock copolymer (PEO-PPO-PEO) to form 125I-MT NPs. The 125I-MT NPs exhibited high labeling efficiency (98 ± 2%) with a hydrodynamic diameter of 91.3 ± 5.5 nm. In vitro and in vivo studies demonstrated that 125I-MT NPs emitted intensive NIR-II fluorescence and SPECT signals, and possessed good biocompatibility. By using a breast tumor xenograft mouse model after intravenous injection of 125I-MT NPs, the SPECT imaging and NIR-II imaging showed clear images of tumor tissues at 8 h and 48 h postinjection, respectively, suggesting the feasibility of using 125I-MT NPs to detect tumors before surgery and visualize the dissection area during surgery. In addition, the SPECT scan of a lymph node mapping was performed at 1 h postinjection and NIR-II fluorescence imaging was carried out at 4 h postinjection. This further guarantees the accurate imaging of lymph nodes before and during surgery for lymphadenectomy. Overall 125I-MT NP is a promising, practical imaging probe for sequential imaging and precision cancer therapy.