A two-photon excitation fluorescence (TPEF) microscopy system under broadband excitation of femtosecond pulses is built in this study. The TPEF spectrum of an eosin stain is recorded, and the TPEF microscopy system is equipped with a suitable detection window matching the TPEF spectrum of the eosin stain. TPEF microscopy is demonstrated using a solid eosin sample and eosin-stained mouse brain sections through detecting the TPEF signal from eosin staining. Three-dimensional (3D) reconstruction of the mouse brain section across its thickness is realized with several TPEF imaging frames acquired via z-axis scanning with a step size of 1 μm. Two-dimensional TPEF imaging frames and 3D reconstruction of the brain section demonstrated high performance of TPEF microscopy in the observation of eosin-stained biological sections.