Screen printed carbon electrodes (SPCEs) are widely used in the field of electrochemical sensors, especially in the areas of food, water, environmental quality, and healthcare applications. In this context, microband SPCEs were fabricated on Whatman paper, overhead projection (OHP) sheet and cotton textile substrates for the development of cost-effective electrochemical biosensors. The fabricated electrodes were subjected to three consecutive processes such as thermal, electrochemical and oxygen plasma treatment. Surface morphologies of SPCEs after each treatment stage were observed using scanning electron microscope for process standardization. Electrochemical performances of the fabricated SPCEs were analyzed using cyclic voltammetry. SPCEs on OHP substrate exhibited a single electron transfer rate at Delta E-p of 57 mV and i(pa)/i(pc) = 1 (n = 3) with significant repeatability and reproducibility compared to SPCEs on paper and textile substrates. The role of activation of edge plane pyrolytic graphite of SPCEs in achieving the reversible reaction with single electron transfer characteristics has been analysed and reported. The effectiveness of the activated SPCEs on the OHP substrate was tested by detecting 1 mM of uric acid and ascorbic acid with KCl as an electrolyte. Graphic abstract