The demand for low-cost, portable, and sensitive analytical devices has fueled the development of 3D-printed biosensors. This study evaluates the effect of silk fibroin incorporation on the electrical properties of graphite-PLA electrodes manufactured via 3D printing. Electrochemical Impedance Spectroscopy (EIS) method was utilized to assess capacitive–resistive behavior under dry conditions, and with PBS buffer, at fibroin concentrations of 0%, 0.04%, 0.4%, and 4%. Fibroin modulated impedance magnitude values without a clear trend, indicating the presence of additional influencing factors. The results promote better understanding of biofunctionalization effects in 3D-printed electrodes and support their potential for biomedical, environmental, and industrial sensing applications.