A new fluorescent imaging probe indocyanine green‐VC (ICG‐VC) in the second near‐infrared window (NIR‐II) was fabricated based on the urokinase‐type plasminogen activator receptor (uPAR) targeted polypeptide VC (VSNKYFSNIHWGC). The relative quantum yield (ΦF)was 0.34 %. The tail of its fluorescence emission wavelength exceeded 1000 nm with excellent photostability. In vitro cell analysis, MTT results indicated the high biocompatibility of ICG‐VC with U87MG cells. ICG‐VC showed good photothermal conversion behavior and photothermal stability.In vivo NIR‐II fluorescence imaging, ICG‐VC exhibited high spatiotemporal resolution in the lymphatic vessels with a high resolution of 717 μm and signal‐to‐noise ratio of 4.36, and displayed an excellent U87MG tumor uptake within 4 h, which reached the maximum at 8 h with a signal‐to‐noise ratio of 5.32.