In this study, malate dehydrogenase (MDH) enzyme was immobilized on a cation octadecylamine (ODA(+)) monolayer at the air-water interface. Pressure-area (pi-A) isotherm studies confirmed that the ODA-MDH system formed a stable monolayer at the air-water interface. The as-prepared MDH-ODA monolayer was transferred onto an indium tin oxide coated glass substrate (ITO) by the Langmuir-Blodgett (LB) method as an MDH-ODA/ITO LB film and characterized using FT-IR, SEM and UV-Vis absorption spectroscopy. The catalytic activity of the enzyme immobilized electrode (MDH-ODA/ITO) was assayed by sensing malic acid (MA) in the range of 10.0-50.0 mM. The high and preserved enzymatic activity of MDH in in vitro media was explored by following the absorbance (A(340 nm)) of nicotinamide adenine dinucleotide (NADH). Moreover, the highly sensitive electrochemical biosensor behavior of the MDA-ODA/ITO electrode for MA detection was displayed by cyclic voltammetry studies. The electrochemical studies revealed that a voltammetric current from the MDA-ODA/ITO electrode was obtained, while the ODA/ITO electrode did not show this current response. The MDA-ODA/ITO demonstrated sensitive electrochemical sensor ability for quantification of MA in both standard solutions and real samples.