Purpose: To visualize and quantify the three-dimensional (3D) spatial relationships of the structures of the aqueous outflow system (AOS) within intact enucleated mouse eyes using spectral two-photon microscopy (TPM) techniques. Methods: Spectral TPM, including two-photon autofluorescence (TPAF) and second-harmonic generation (SHG), were used to image the small structures of the AOS within the limbal region of freshly enucleated mouse eyes. Long infrared excitation wavelengths (930 nm) were used to reduce optical scattering and autofluorescent background. Image stacks were collected for 3D image rendering and structural segmentation. For anatomical reference, vascular perfusion with fluorescent-conjugated dextran (150 KDa) and trans-corneal perfusion with 0.1 μm fluorescent polystyrene beads were separately performed to identify the episcleral veins (EV) and the trabecular meshwork (TM) and Schlemm's canal (SC), respectively. Results: Three-dimensional rendering and segmentation of spectral two-photon images revealed detailed structures of the AOS, including SC, collector channels (CC), and aqueous veins (AV). The collagen of the TM was detected proximal to SC. The long and short axes of the SC were 82.2 ± 22.2 μm and 6.7 ± 1.4 μm. The diameters of the CC averaged 25.6 ± 7.9 μm where they originated from the SC (ostia), enlarged to 34.1 ± 13.1 μm at the midpoint, and narrowed to 18.3 ± 4.8 μm at the junction of the AV. The diameter of the AV averaged 12.5 ± 3.4 μm. Conclusions: Spectral TPM can be used to reconstruct and measure the spatial relationships of both large and small AOS structures, which will allow for better understanding of distal aqueous outflow dynamics.