Gold nanoparticles (AuNPs) were synthesized and stabilized using ecological strategies: the extracts of the leaves of the plants Gliricidia sepium (GS) and Petiveria alliacea (PA) reduced the metallic Au ions to AuNPs. The AuNPs were analyzed as surface-enhanced Raman scattering (SERS) substrates for pyridoxine detection (vitamin B6). UV–vis spectroscopy was carried out to assess the stability of the AuNPs. As a result, absorption bands around 530 and 540 nm were obtained for AuNPs-PA and AuNPs-GS, respectively. Both cases associated it with localized surface plasmon resonance (LSPR). In the final stage of the synthesis, to stabilize the AuNPs, carboxymethyl cellulose (CMC) was added; however, LSPR bands do not exhibit bathochromic or hypsochromic shifts with the addition of CMC. Transmission electron microscopy (TEM) micrographs show relatively spherical morphologies; the particle diameters were detected around 7.7 and 12.7 nm for AuNPs-PA and AuNPs-GS, respectively. The nanomaterials were evaluated as SERS substrates on pyridoxine, revealing an intensification in the vibrational mode centered at 688 cm–1 associated with the pyridinic ring. Complementarily, different density functional theory functionals were included to obtain molecular descriptors on the Aun-cluster-pyridoxine interaction to study the SERS behavior.