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.