An innovative electrochemical interface for quantitation of L-proline (L-Pro) based on ternary amplification strategy was fabricated. In this work, gold nanoparticles prepared by soft template methodology were immobilized onto green and biocompatible nanocomposite containing poly as a conductive matrix and graphene quantum dots as the amplification element. Therefore, a novel multilayer film based on poly-L-cysteine, graphene quantum dots (GQDs), and gold nanoparticles (GNPs) was exploited to develop a highly sensitive electrochemical sensor for the detection of L-Pro. Fully electrochemical methodology was used to prepare a new transducer on a glassy carbon electrode, which provided a high surface area towards sensitive detection of L-Pro. The prepared electrode was employed for the detection of L-Pro. Under optimized conditions, the calibration curve for L-Pro concentration was linear in 0.5 nM - 10 mM with a low limit of quantification of 0.1 nM. The practical analytical utility of the modified electrode was illustrated by determination of L-Pro in unprocessed human plasma samples.