The peripheral nervous system (PNS) is essential for performing and maintaining various motor and sensory functions. Abnormalities can lead to a series of peripheral neurological conditions, such as paraesthesia, pain, or spasms, which are debilitating and lowering the quality of life. The current guidelines for diagnosis rely predominantly on clinical symptoms resulting from PNS dysfunction, which occur already at an advanced stage. There are currently no effective methods that visually reflect the extent of peripheral neuropathy. In our study, we present a novel in vivo and in situ real-time imaging of peripheral nerves based on the second near-infrared window (NIR-II) fluorescence. In NIR-II system, PbS Qds with NIR-II fluorescence specifically bound to motor neuron-specific protein agrin, acting as image contrast. In mice model, peripheral nerves were visible as soon as after 2 h post injection. We provide evidence for the efficacy of this approach, which allows to directly demonstrate peripheral nerves, their structure, and potential damage sites and degree. Furthermore, our products were of good biocompatibility, while the neural fluorescence signal was solid, bright and stable for 4 h in vivo. Thus, overall, our results suggest that NIR-II is an effective new method for direct imaging of peripheral nerves in vivo, opening new horizons on early, improved and more precise, targeted diagnosis. A resulting more rapid installation of personalized therapy facilitates a better prognosis of clinical peripheral neuropathy.