Lactose (LAC) is the main disaccharide sugar found in milk and dairy products, making it easily accessible as a food source. In addition to its importance for human and animal health, LAC concentrations serve as a biomarker of milk quality. In this study, we reported the detection of LAC using a highly sensitive electrochemical sensor based on rhodamine B (RhB) dye. The prepared RhB-based sensor exhibited excellent characteristics, including a straightforward manufacturing technique, cost-effectiveness, and exceptional sensitivity. We conducted a comparative analysis of the electrochemical response of the novel RhB-based sensor, evaluating its electrochemical performance across varying LAC concentrations and when different analyte materials (glucose, fructose, and maltose) were employed. The proposed analytical method was utilized to determine LAC using an electrochemical RhB-based sensor under optimal experimental conditions. As a result, it was experimentally observed that high sensor sensitivity of RhB based sensor against LAC was inversely proportional to the impedance value and directly proportional to conductance which corolated with the transport mechanism of RhB.