Ascorbic acid is implicated in various diseases such as scurvy, oxidative stress, cardiovascular diseases, etc. Herein, for the first time, a simple and efficient strategy was used to synthesize cross-linked chitosan-stabilized copper oxide nanoparticles (CuO@C-CS) as a non-toxic and biodegradable-based approach. Various spectroscopic techniques, including FTIR, XRD, SEM, EDX, TGA, and elemental mapping, confirmed the synthesis of the material. The synthesized nanozyme (CuO@C-CS) was used as a peroxidase mimic for the detection of ascorbic acid, through the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) with the assistance of hydrogen peroxide. The synthesized mimic enzyme transforms colorless TMB into oxTMB. The sensing of ascorbic acid was achieved through the peroxidase-like inhibitory activity of the mimic enzyme along with the reduction of oxTMB. The sensor system was fine-tuned, and it showed a limit of detection, a limit of quantification, a linear range, and regression coefficient values of 0.24 μM, 0.80 μM, 1-96 μM, and 0.999, respectively. The fabricated sensor was very selective in the presence of various potential interferents. The proposed sensor was successfully applied to commercially available orange juices for the qualitative and quantitative determination of ascorbic acid. The sensor can be used for the determination of ascorbic acid in biomedical and food samples.