The use of colloidal nanoparticles suffers from the drawbacks of potential color interference and substrate-induced aggregation. To overcome the limitations, a catalyst was developed by crosslinking Cu-doped carbon dots (Cu-CDs) with chitosan. Cu-CDs with high peroxidase activity were prepared by using a rapid microwave-assisted method. The Cu-CDs containing 6.88% of Cu had an average particle size of 2.25 nm and exhibited 9% of fluorescence quantum yield. The nanozyme/film composite was prepared by crosslinking between the amino groups of Cu-CDs and those of chitosan via a glutaraldehyde linker. A HO-mediated tetramethylbenzidine (TMB) oxidation reaction was use to evaluate the peroxidase activity of the film. Based on the TMB color changes, colorimetric assays were developed for the detection of HO and glucose at an absorption wavelength 652 nm. Under the optimal conditions, the linear ranges for HO and glucose were 0.625-40 µM and 1.9-125 µM, respectively, and the detection limits were 0.12 µM and 0.69 µM, respectively. The colorimetric assay was also applied to analyze diluted human serum samples spiked with glucose. Furthermore, this biodegradable, non-toxic, and easy-to-handle nanozyme composite could be stored for over 4 weeks without a significant decrease in activity.