Although microRNAs are considered key regulators of plant responses to biotic stresses, their accurate measurement is challenging due to their extremely low concentrations. The aim of this study was to develop and optimize a microRNA-based electrochemical biosensor and to evaluate its performance in detecting biotic stress caused by spot blotch disease in barley plants. An electrochemical biosensor was designed based on the hybridization of the target microRNA with an oligonucleotide probe immobilized on a gold electrode surface, followed by the adsorption of positively charged silver–polyethyleneimine nanoparticles. Support vector machine regression was employed to optimize the probe volume and nanoparticle mass. The experimental dataset consisted of 100 observations, and five-fold cross-validation was used to evaluate model performance. After confirming the satisfactory performance of the model in predicting the electrochemical response of the biosensor (mean squared error of 0.004 and coefficient of determination of 0.97), the optimal probe volume and nanoparticle mass were determined by the developed model to be 9.15 mL and 1.85 µg, respectively. The calibration curve of the optimized biosensor exhibited a linear range from 10⁻19 to 10⁻11, and the biosensor was used to measure the concentrations of miRNA168 and miRNA397, which play essential roles in plant responses to spot blotch fungal disease. The biosensor data indicated a significant difference in the concentrations of these microRNAs between healthy and infected plants, confirming its effectiveness for early disease detection. Overall, the results demonstrate the considerable potential of the proposed biosensor for accurate identification of biotic stresses in plants.