To overcome the disadvantage of simple bimetallic nanocluster solutions being difficult to store and utilize, we prepared and obtained a novel gold and copper bimetallic nanocluster-doped chitosan fluorescent composite film. In this study, gold and copper bimetallic nanoclusters emitting strong red fluorescence were first synthesized by a chemical reduction method. Subsequently, a novel gold and copper bimetallic nanocluster-doped chitosan fluorescent composite film was successfully prepared by a solution casting method. After 60 minutes of UV light irradiation or 30 days at room temperature, the relative fluorescence intensity values of the composite film decreased by 0.9% and 1.2%, respectively. This indicates that its optical properties are stable and that it can be stored for a long time. The composite film has strong and bright red fluorescence and can be used as a fluorescent probe to achieve real-time detection of Cr(VI). It also has a low detection limit for Cr(VI) (0.26 ppb), so it can be applied to the detection of Cr(VI) in actual water samples and get satisfactory detection results. Due to its portability, high selectivity, and high sensitivity, it can also be extended to chemical and food detection.