Histamine, a significant biogenic amine produced during the storage and processing of meat products, serves as a key indicator for evaluating food freshness and safety. To meet the demand for rapid and sensitive detection of histamine in meat, an electrochemical biosensor based on a composite of diamine oxidase (DAO) and a graphitic carbon nitride/titanium dioxide (g-C3N4/TiO2) heterojunction was developed. The g-C3N4/TiO2 heterojunction material was prepared via a one-step calcination method and systematically characterized using techniques such as scanning electron microscopy, transmission electron microscopy, and Fourier-transform infrared spectroscopy. These analyses confirmed the uniform loading of TiO2 nanoparticles onto the g-C3N4 sheets and the formation of a stable heterojunction interface. Density functional theory calculations further verified at the atomic scale that this heterojunction interface effectively enhances histamine adsorption and promotes interfacial charge transfer. Building on the advantages of the heterojunction elucidated by theoretical calculations, the sensor improves enzyme immobilization efficiency and establishes an efficient electron transfer pathway, thereby achieving effective conversion and amplification of biological recognition signals into electrochemical signals. The results showed that the biosensor exhibited a good linear response in the range of 0.05–9.0 mmol/L, with a detection limit of 0.226 μmol/L (S/N = 3), and the interference-induced signal deviation was less than 8%. In practical meat sample analysis, the relative standard deviation was below 5%, indicating good accuracy and reproducibility. In conclusion, the constructed g-C3N4/TiO2 biosensor is suitable for rapid screening and quantitative detection of histamine in meat products, providing a stable and reliable approach for histamine monitoring in food safety applications.