Optical coherence tomography (OCT) imaging mainly uses backscattered light to visualize the structural and functional information on biological tissues. In particular, OCT angiography can not only map the capillary networks but also capture the blood flow in the tissue microenvironment, making it a good candidate for neuroimaging and tumor imaging in vivo and in real time. To further improve the detection accuracy of cancer or brain disorders, it is essential to develop a natural and nontoxic contrast agent for enhanced OCT imaging in the second near-infrared (NIR-II) window. In this study, a superior biocompatible and highly scattering NIR-II fat nanoemulsion was constructed to improve OCT imaging contrast and depth for monitoring the vascular network changes of the cerebral cortex or tumor. In vivo experimental results demonstrated that a natural fat nanoemulsion can serve as an excellent probe for enhanced OCT neuroimaging and tumor imaging.