Three-dimensional (3D) printing technology offers attractive possibilities for many fields. In electrochemistry, 3D printing technology has been used to fabricate customized 3D-printed electrodes as a platform to develop bio/sensing, energy generation and storage devices. Here, we use a 3D-printed graphene/polylactic (PLA) electrode made by additive manufacturing technology and immobilize horseradish peroxidase (HRP) to create a direct electron transfer enzyme-based biosensors for hydrogen peroxide detection. Gold nanoparticles are included in the system to confirm and facilitate heterogeneous electron transfer. This work opens a new direction for the fabrication of third-generation electrochemical biosensors using 3D printing technology, with implications for applications in the environmental and biomedical fields.