Glycine is an essential amino acid involved in synthesizing a variety of important biomolecules, and its concentration can influence numerous biochemical processes, including the severity of symptoms in a wide range of conditions in humans, such as cancer, schizophrenia, major depression, and diabetes. While a few costly or labour-intensive methods are currently available, we have developed a new enzymatic biosensor that can accurately measure glycine levels with remarkable simplicity. By employing immobilized laccase enzymes in combination with a surface plasmon resonance (SPR) device, our system achieved a limit of detection (LOD) of 9.95 mM and a limit of quantification (LOQ) of 33.19 mM. In addition, it demonstrated a recovery rate of 97.64 ± 7.71%. Moreover, the biosensor maintained consistent signal intensity over 21 days and supported a total of 60 analyses using the same immobilized enzyme setup, demonstrating excellent reusability. Notably, this study marks the first time glycine has been determined using an enzymatic SPR-based platform.