The adsorption and photo-Fenton degradation of tetracycline (TC) over Fe saturated nanoporous montmorillonite was analyzed. The synthesized samples were characterized using XRD, FTIR, SEM, and XRF analysis, and the adsorption and desorption of TC onto these samples as well as the antimicrobial activity of TC during these processes were analyzed at different pH. The results indicated that the montmorillonite is a great adsorbent for the separation of the TC from aqueous solutions, however, after increasing the amount of TC adsorbed, the desorption process started, and up to 50% of TC adsorbed onto non-modified montmorillonite was released back to the solution with almost no changes in its antimicrobial activity. After acid treatment (for creation of nanoporous layers) and Fe saturation of the montmorillonite, almost similar great separation was achieved compared to non-modified montmorillonite. In addition, the desorption of TC from modified montmorillonite was still high up to 40% of adsorbed TC. However, simultaneous adsorption and photodegradation of TC were detected and almost no antimicrobial activity was detected after 180 min of visible light irradiation, which could be due to the photo-Fenton degradation of TC on the modified montmorillonite surface. In the porous structures of modified montmorillonite high ˙OH radicals were created in the photo-Fenton reaction and were measured using the Coumarin technique. The ˙OH radicals help the degradation of TC as proposed in an oxidation process. Surprisingly, more than 90 % of antimicrobial activity of the TC decreased under visible light (after 180 min) when desorbed from nanoporous Fe-saturated montmorillonite compared to natural montmorillonite. To the best of our knowledge, this is the first time that such a high TC desorption rate from an adsorbent with the least remained antimicrobial activity is reported which makes nanoporous Fe-saturated montmorillonite a perfect separation substance of TC from the environment.