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Abstract: A highly selective nitric oxide (NO) biosensor was developed by immobilizing microperoxidase (MP) onto the MWCNT-poly-5,2′:5′,2″-terthiophene-3′-carboxylic acid (PTTCA) nanocomposite. Catalase (CAS) and superoxide dismutase (SOD) co-immobilized on the probe successfully protected the interferences of H2O2 and O2 − during NO detection. The nanocomposite layer showed the direct electron transfer processes of the immobilized CAS, SOD, and MP simultaneously at −0.11/+0.04, −0.33/−0.27, and −0.47/−0.40V vs. Ag/AgCl. Au nanoparticles (AuNPs) were electrodeposited on a glassy carbon surface to enhance the sensitivity of the sensor probe. The layers of CAS/SOD/MP/MWCNT-PTTCA/AuNPs were characterized using SEM, XPS, and QCM. The CAS/SOD/MP/MWCNT-PTTCA/AuNPs probe showed an excellent performance in the electrocatalytic reduction of NO which was attributed to the heme group of MP. Experimental conditions affecting the sensitivity of the biosensor were optimized in terms of pH, temperature, and applied potential. The dynamic range of NO analysis was from 1.0 to 40μM with the detection limit of 4.3±0.2nM. The reliability of biosensor was examined for the determination of NO released from the real samples of rat liver, stomach (AGS), and intestinal (HT-29) cancer cells. [ABSTRACT FROM AUTHOR]