A novel conductive composite film containing functionalized multi-walled carbon nanotubes (f-MWCNTs) with poly (neutral red) (PNR) was synthesized on glassy carbon electrodes (GC) by potentiostatic method. The composite film exhibited promising electrocatalytic oxidation of mixture of biochemical compounds such as ascorbic acid (AA), dopamine (DA) and uric acid (UA) in pH 4.0 aqueous solutions. It was also produced on gold electrodes by using electrochemical quartz crystal microbalance technique, which revealed that the functional properties of composite film were enhanced because of the presence of both f-MWCNTs and PNR. The surface morphology of the polymer and composite film deposited on transparent semiconductor tin oxide electrodes were studied using scanning electron microscopy and atomic force microscopy. These two techniques showed that the PNR was fibrous and incorporated on f-MWCNTs. The electrocatalytic responses of neurotransmitters at composite films were measured using both cyclic voltammetry (CV) and differential pulse voltammetry (DPV). These experiments revealed that the difference in f-MWCNTs loading present in the composite film affected the electrocatalysis in such a way, that higher the loading showed an enhanced electrocatalytic activity. From further electrocatalysis studies, well separated voltammetric peaks were obtained at the composite film modified GC for AA, DA and UA with the peak separation of 0.17 V between AA–DA and 0.15 V between DA–UA. The sensitivity of the composite film towards AA, DA and UA in DPV technique was found to be 0.028, 0.146 and 0.084 μA μM−1, respectively.