Oxygen and glucose biosensors have been designed, fabricated, characterized and optimized for real-time continuous monitoring on a new smart catheter for use in patients with traumatic brain injury (TBI). Oxygen sensors with three-electrode configuration were designed to achieve zero net oxygen consumption. Glucose sensors were based on the use of platinum nanoparticle-enhanced electrodes that were modified with polycation and glucose oxidase immobilized by chitosan matrix. An iridium oxide electrode was developed to work as a biocompatible reference electrode with enhanced durability and stability in the biological solutions. A study of the effect of temperature on oxygen sensor performance, and both temperature and oxygen effects on glucose sensor performance were accomplished to enhance their operative stability and provide useful information for in vivo applications. A new methodology for automatic correction of the temperature and oxygen dependence of biosensor outputs is demonstrated through programmed LabView software. In vitro experiments in both physiological and pathophysiological ranges (oxygen: 0-60 mmHg; glucose: 0.1-10 mM; temperature: 25-40 degrees C) with clinical samples of cerebrospinal fluid obtained from TBI patients have demonstrated stable measurements with enhanced accuracy, indicating the feasibility of the sensors for a real-time continuous in vivo monitoring.