The use of skin flaps to fill large defects is a key surgical technique in reconstructive surgery, effective real-time in vivo imaging for flap design and use is urgent. Currently, fluorescent imaging in the second NIR window (NIR-II; 1000-1700 nm) is characterized by non-radiation, less expensive and higher resolution in comparisons with the first NIR window (NIR-I; 700-900 nm) and other traditional imaging modalities. In this article, we identified the location and numbers of perforators and choke zone via NIR-II imaging. Then, eight abdominal perforator flaps were established and the perfusion zones were evaluatedat special time points. Finally, after eight pedicled flaps establishment, NIR-II imaging was used to guide the optimal timing for division of flap pedicle. The results showed that NIR-II fluorescence imaging with indocyanine green (ICG) can reliably visualize vascular supply, which makes it to be an accurate and in vivo imaging approach to flap clinical design and use.