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Ascorbic acid (AA) determination is of high importance in the diagnosis and treatment of diseases. Herein, we report a new ratiometric electrochemical sensor for ascorbic acid using a glassy carbon electrode (GCE) modified with a copper nanoparticle@resin nanosphere nanocomposite (CuNPs@RNS). The ratiometric strategy was established by immobilizing an internal reference (thionine) on the modified electrode using cyclic voltammetry (CV). The performance of the modified electrodes as well as the newly established ratiometric strategy was explored. The sensing platform had good electrocatalytic ability, reproducibility, and stability. In addition, the ratiometric strategy significantly improved the performance of electrochemical sensing with a wide linear range (0.0837 to 15.5 μM) and a low detection limit of 0.0279 μM under the optimal conditions. The original ratiometric electrochemical sensor was successfully applied to monitor ascorbic acid in tablets and urine. These results showed that the sensor provides a new strategy and broad prospects for biomolecular sensing with reliability and high sensitivity. [ABSTRACT FROM AUTHOR]