Research and development of materials and strategies for real-time, amperometric sensors and biosensors with potential in-vitro or in-vivo applications for clinically relevant physiological agents continues to be a relevant scientific endeavor. Within this field of study, a large number of reports continue to focus on enzyme-based glucose detection because it serves both as a well-understood fundamental model system and as a clinically relevant sensing goal for diabetics. It is, however, relatively rare that the same strategies and materials utilized for glucose sensing prove to be robust and versatile enough to be readily adapted to different target molecules with clinical relevance. It follows then that a significant achievement would be the demonstration of a strategy and the use of materials that offer this type of versatility while maintaining superior performance toward a molecule with bioanalytical implications and possible medical applications. Our work explores layer-by-layer (LbL) strategies for amperometric biosensor and sensor designs, where each layer or material within the composite film, including the incorporation of different nanomaterials, is functional with respect to sensing sensitivity and/or selectivity. Additionally, this research also involves sensor development toward practical and effective clinical usage where the LbL strategies must be successfully adapted and miniaturized to needle or wire electrodes that can potentially function in vitro as a bedside device, within catheters for continuous, real-time measurements, or as an in vivo implant operating in biological media and physiologically relevant concentrations. Fundamental studies proceed with a glucose model system before adapting the strategy and materials to specifically targeted clinical measurements including early detection/monitoring for sepsis, pregnancy-induced hypertension, and prostate cancer among others.