Formulations for the total fluorescence intensity of fluorescent microspheres in slabs of cardiac tissue were determined experimentally and theoretically. The tissue depth, at which the slab can be considered as a semi-infinite turbid medium, and critical layer thickness, which accounts for the most emission intensity were evaluated to be 8–9 and 3–5 mm, respectively, for the cardiac tissue. When fluorescent microspheres are linearly distributed across the slab depth, the mean absorption of them is proportional to the sum of their normalized total emissions in the slab excited from both sides. The formulations may be used for the fluorescence images analysis of cardiac and other biological tissues.