Ultrafine plasma jet is a promising technology with great potential for nano- or micro-scale surface modification. In this letter, we demonstrated the use of ultrafine atmospheric pressure plasma jet (APPJ) for patterning bio-immobilization on vertically aligned carbon nanotube (CNT) microarray platform without a physical mask. The biotin-avidin system was utilized to demonstrate localized biomolecule patterning on the biosensor devices. Using ±7.5 kV square-wave pulses, the optimum condition of plasma jet with He/NH3 gas mixture and 2.5 s treatment period has been obtained to functionalize CNTs. The functionalized CNTs were covalently linked to biotin, bovine serum albumin (BSA), and avidin-(fluorescein isothiocyanate) FITC, sequentially. BSA was necessary as a blocking agent to protect the untreated CNTs from avidin adsorption. The localized patterning results have been evaluated from avidin-FITC fluorescence signals analyzed using a fluorescence microscope. The patterning of biomolecules on the CNT microarray platform using ultrafine APPJ provides a means for potential application of microarray biosensors based on CNTs.