Fluorescence imaging in the second near-infrared window (NIR-II, 1000–1700 nm) is a promising modality for real-time imaging of cancer and image-guided surgery with superior in vivo optical properties. So far, very few NIR-II fluorophores have been reported for in vivo biomedical imaging of chemically-induced spontaneous breast carcinoma. Herein, a NIR-II fluorescent probe CH1055-F3 with the nucleolin-targeted tumor-homing peptide F3 was demonstrated to preferentially accumulate in 4T1 tumors. More importantly, CH1055-F3 exhibited specific NIR-II signals with high spatial and temporal resolution, strong tumor uptake, and remarkable NIR-II image-guided surgery in dimethylbenzanthracene (DMBA)-induced spontaneous breast tumor rats. This report presents the first tumor-homing peptide-based NIR-II probe to diagnose transplantable and spontaneous breast tumors by the active targeting.