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.