Traumatic penumbra (TP) is a region with recoverable potential around the primary lesion of brain injury. Rapid and accurate imaging for identifying TP is essential for treating traumatic brain injury (TBI). In this study, we first established traumatic brain injuries (TBIs) in rats using a modified Feeney method, followed by label-free imaging of brain tissue sections with multiphoton fluorescence microscopy. The results showed that the technique effectively imaged normal and traumatic brain tissues, and revealed pathological features such as extracellular matrix changes, vascular cell proliferation, and intracellular edema in the traumatic penumbra. Compared with normal brain tissue, the extracellular matrix in the TP was sparse, cells were disorganized, and hyperplastic vascular cells emitted higher two-photon excited fluorescence (TPEF) signals. Our research demonstrates the potential of multiphoton fluorescence technology in the rapid diagnosis and therapeutic evaluation of TBI.