This paper reports the novelty of the synthesis and characterization of an eco-friendly, cost-effective, and easily handled graphene-based nanocomposite blended with PANI and ZnO to be used as a sensor for detecting the amino acid. Graphene oxide, ZnO, PANI, and PANI-ZnO-rGO nanocomposite samples were synthesized by using different preparation techniques such as Hummer's method, precipitation method, chemical oxidative polymerization method, and probe sonication, respectively. The samples were characterized by FTIR, XRD, UV-Vis, PL, and FMSEM techniques to confirm the structural, optical, and morphological properties of the samples. FTIR results show that PANI spectra interacted with rGO-ZnO through its characteristic band (N-H) at 3234 cm-1, indicating the formation of the PANI-ZnO-rGO nanocomposites. In addition, XRD results reveal the characteristic peaks of the PANI sample at 25 degrees and the rGO-ZnO nanocomposite at 34.3 degrees, 36.3 degrees, 47.6 degrees, 56.6 degrees, 62.7 degrees, and 68 degrees, respectively, which confirm the existence of the combination between PANI and rGO-ZnO. The nanostructure nature of the composite was confirmed by the calculated nanoscale values of the average crystallite size and FESEM micrographs. Moreover, UV-Vis absorption spectra show evidence of the formation of the PANI-ZnO-rGO nanocomposites, whereas a sharp peak at 326 nm, followed by a broad peak at 633 nm, which is a blue-shifted absorption peak of PANI-ZnO-rGO composites at 676 nm. Furthermore, PL spectra confirming the prepared nanocomposite has a significant selectivity to L-Arginine. These results confirm that the PANI- ZnO-rGO nanocomposites make strong changes to the optical properties of L-Arginine, indicating that the PANI-ZnO-rGO nanocomposite is a candidate for biosensors.