In this work, electrochromic Prussian blue (PB) films were electrodeposited on the surface of indium tin oxide (ITO) electrodes, and a dispersion mixture of glucose oxidase (GOD), chitosan (CS) and gold nanoclusters (AuNCs) was then cast on the PB surface to form CS-AuNC-GOD/PB nanocomposite film electrodes. The blue PB component in the films could be changed into its colourless reduced form of Prussian white (PW) upon application of −0.2 V. The addition of glucose to the solution would produce H₂O₂ with the help of GOD in the films and oxygen in the solution, which could oxidize PW back to PB. In the meantime, the fluorescence emission signal of the AuNCs in the films was greatly influenced by the form of PB/PW. Based on these properties, the amperometric current, fluorescence intensity and UV-vis absorbance of the film electrodes demonstrated potential- and glucose-sensitive ON–OFF behaviors. Thus, a 2-input/3-output biomolecular logic gate system with 3 different types of output signals and a 2-to-1 encoder were developed. Furthermore, a resettable and reprogrammable 3-input biomolecular keypad lock was established with fluorescence intensity and UV-vis absorbance as dual outputs, which greatly enhanced the security level of the keypad lock. This work reported for the first time an enzyme-based keypad lock with dual outputs, which might open a new avenue to design more complicated biomolecular keypad lock systems.