Transferred multi-walled carbon nanotube (MWCNT)-modified platinum thin-film immunosensing electrode material was engineered on a glass substrate and fabricated a fully-integrated electrochemical three-electrode system for monitoring Legionella pneumophila. The transferred MWCNT film was treated with oxygen plasma to improve its electrochemical response and electrical conductivity. We voltammetrically characterized and optimized the electrochemical performance of the fabricated electrode for direct detection of Legionella pneumophila-specific peptidoglycan-associated lipoprotein (PAL) and maltose binding protein (MBP) peptidoglycan-associated lipoprotein (MBP-PAL) fusion. The latter, as an intermediate product to yield the former, has important roles in the growth and purification of PAL, which commercial enzyme-linked immunosorbent assay (ELISA) kits require as a target substrate. Consequently, direct electrochemical detection of MBP-PAL compared to PAL by square-wave voltammetry showed a greater than 50% increase in sensitivity with a lower detection limit of 5?pg?mL-1. We also investigated the affinity properties by determining kinetic parameters of the PAL and the MBP-PAL in relation to polyclonal antibodies immobilized on transferred MWCNT substrates using MichaelisMenten assumptions and a HanesWoolf plot. This new method presented herein could save the time and effort for the separation and purification of PAL form MBP-PAL fusions that are required for performing ELISA-based immunoassay. Biotechnol. Bioeng. 2012; 109:14711478. (c) 2011 Wiley Periodicals, Inc.