The dual-axis confocal (DAC) configuration provides a high axial resolution, long working distance (WD), and large dynamic range. These properties can reveal depth-resolved fluorescence spectra. We present a depth sensitive fluorescence spectroscopy based on the DAC configuration. The system enables high axial resolution of 3.23 µm and a long WD of 3.73 mm compared to that of 4.68 µm and 2.1 mm for comparable single-axis confocal configurations, respectively. Besides, a DAC configuration also offers a superior dynamic range and rejection of out-of-focus scattered light based on the principle of Huygens-Fresnel integrals. Additionally, to locate the target layer, the collection path of the DAC configuration will be used as the other illumination path, forming a dual-axis illumination configuration. These beam paths are used to locate the target layer using a white light imaging system with a commercial low numerical aperture objective. A multi-layer fluorescence phantom of Barrett’s esophagus containing fluorescein isothiocyanate and Alexa Fluor 514 was used to verify the principle of depth-resolved fluorescence spectroscopy. The results show that the DAC configuration can collect fluorescence spectra from microscopic regions with high axial resolution.