Abstract: Periphytons are the micro-organisms growing on stones, sticks aquatic macrophytes, and submerged surfaces. Periphyton is the easiest source of food for fishes in an ecosystem. In the present study different structures of cotton stems and cotton flowers were evaluated for the periphyton production in the laboratory. The physico-chemical properties of substrates added water and initial water was analyzed by standard methods. The results revealed that the biomass of periphyton was high on the isolated cotton stems than the other substrates. The taxonomic composition of periphyton included, Chlorophyceae (green Algae), Bacillariophyceae (Diatoms), Arthropods (mosquito larvae), Ostracoda (Cypris sp.) and Copepods (Cyclop sp.) and Mollusca (Lymnae sp.). The Chlorophyceae and Bacillariophyceae groups were dominant in all substrates except cotton flower. The physico-chemical properties of water were suitable for the periphyton production. Thus, among the all substrates, the isolated cotton stem was comparatively more efficient for periphyton development. The isolated cotton stems could be utilized in the periphyton based aquaculture systems, which is eco-friendly and sustainable technology