© 2020 Wiley-VCH GmbH Dysregulated microRNA associated pathways contribute to the pathology of neurological disorders, hence presenting themselves as a potential candidate for motor neuron disease (MND) diagnosis. Herein, we reported an enzymatic amplification-free approach for the electrochemical detection of exosomal microRNA (miR-338-3p) from preconditioned media of motor neurons obtained from amyotrophic lateral sclerosis (ALS) patients and healthy controls. Our assay utilizes a three-step strategy that involves i) initial isolation and purification of exosomal miR-338-3p from patients and healthy controls using biotinylated complementary capture probe followed by heat-release of the specific target, ii) direct adsorption of target miR-338-3p onto the gold-loaded ferric oxide nanocatalyst (AuNP-Fe2O3NC) through affinity interaction between microRNA and exposed gold surfaces within the AuNP-Fe2O3NC, and iii) gold nanocatalyst-induced electrocatalytic signal amplification through methylene blue-ferricyanide redox cycling (MB/[Fe(CN)6]3−). The electrocatalytic signal is monitored by using chronocoulometry at the AuNP–Fe2O3NC-modified screen-printed carbon electrode (AuNP-Fe2O3NC/SPCE). We demonstrated the detection of miR-338-3p as low as 100 aM in spiked buffer samples with a relative standard deviation of (%RSD) <5.0 % (n=5). We also demonstrate the successful detection of miR-338-3p from a small cohort of preconditioned media of motor neurons obtained from ALS patients and healthy controls. The sensor avoids the use of conventional recognition and transduction layers in hybridization-based electrochemical miRNA biosensors, polymerase-based amplifications. It is robust, fast (<2.5 h) and potentially applicable to a wide variety of RNA biomarker detection.