AbstractTwo‐photon microscopy (2PM) has become a gold standard for deep‐tissue observations in the living animal as well as on thick samples. Using 2PM, the endofluorescence properties of biomolecules have shown an interesting potential for the imaging of tissues without any staining. In this short communication, we report a method to observe the different layers of mouse small intestine explants with subcellular resolution and without any staining or clearing. This method allows rapid observations of samples with little to no preparation thanks to the endofluorescence properties of biomolecules such as NAD(P)H or flavins and second‐harmonic generation. Finally, we show different three‐dimensional reconstructions of the mouse small intestine anatomy obtained with this approach to show the potential of this method in morphological studies.