The bio-mediated nanomaterials have expected growing responsiveness due to an increasing requirement to develop naturally nonthreatening technologies in nanomaterial synthesis. Biotic ways to prepare nanomaterials through extracts from the plant (includes stems, leaves, flowers, and roots) and microorganisms were recommended as likely replacements for physical and chemical routes due to their solvent medium and environment eco-friendliness and nontoxicity. This chapter focuses on electrocatalyst prepared by various bio-mediated synthetic ways and used as a green and eco-friendly electrocatalyst to recognize extensive chemical and biologically essential molecules with improved selectivity and sensitivity with low detection limit. The bio-mediated nanocomposite formation processes and their unique properties surface functionalization and electron transfer mechanism discussed in connection with the design and fabrication of sensors. As a final point, the encounters and prospects in developing bio-mediated nanomaterials-based electrochemical sensing technology was outlined.