How to develop near-infrared second window (NIR-II, 1000-1700 nm) fluorescent nanoprobes with a uniform size, strong fluorescence signal and good biosafety owns great clinical requirement. Herein we reported that a two photon fluorescent nanoprobe was developed via encapsulating NIR-II-fluorescent molecules into DSPE-PEG, which was effectively endocytosized by cancer cells, and achieved strong NIR-II fluorescence imaging in cancer cells and cancer cell-beard mice models. Prepared NIR-II-fluorescent nanoprobe exhibited rapid metabolism and excellent biocompatibility. In conclusion, the prepared two photon nanoprobe owns good biosafety, and clinical translational prospect in NIR-II fluorescent imaging of tumour in vivo in near future.