Nitric Oxide (NO) is an important, rapid assay biomarker of many known diseases, including asthma, chronic obstructive pulmonary disease (COPD) as well as other respiratory infections, septic shock, Parkinson’s diseases and Alzheimer’s dementia. Therefore, the detection and measurement of NO in a physiological fluid, either in gas or in liquid phase, are scientifically and clinically significant. The objective of this research is to develop a single-use, disposable cost effective in vitro NO biosensor which can measure NO of ppb (parts per billion) level in gas phase and sub-µM (sub micro mole) level in liquid phase. This biosensor is a thin film platinum based electrochemical sensor. Ni (II) tetrakis (3-methoxy-4-hydroxyphenlyl) porphyrin is mixed with diethylene glycol and Nafion forming a NO selective membrane-electrolyte. Reactive sputtering, laser ablation, thick film printing technologies are used to fabricate the biosensor on a roll-to-roll cost effective processing. Differential pulse voltammetry (DPV) is employed as the measurement technique. The results of this NO biosensor show that this biosensor can measure NO down to 5ppb in gas phase and 0.00242µM of NO in PBS solution (liquid phase). Fabrication and test procedures of this NO biosensor are described in this presentation. In conclusion, this development is scientifically successful and is described as sensor for nitric oxide detection WO2015143197 (2015) by World Intellectual Property Organization on September 24, 2015.