With the help of a hydrothermal approach in this study, we could provide flower-like nanostructures (NSs) of zinc oxide (ZnO) doped with Tb (FL-NS Tb3+/ZnO). Then, FL-NS Tb3+/ZnO morphology was investigated by energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), X-ray powder diffraction (XRD), and map analysis. The results revealed higher activity centers and porosity of this nanocomposite, which were followed by acceptable electrochemical function. Hence, it can be utilized for fabricating an electrochemical sensor with an appropriate response for the simultaneous determination of kynurenic acid (KYN) and tryptophan (TRP). However, as compared with the modified carbon paste electrode (FL-NS Tb3+/ZnO/CPE), the bare carbon paste electrode (BCPE) exhibited a weak response toward KYN and TRP but the modified electrode was followed by a high current response for KYN and TRP at a potential 0.35 and 0.809 V. Therefore, cyclic voltammetry (CV) was applied in optimal experimental conditions to study the electrochemical behaviors of KYN and TRP over the surface of the proposed modified electrode. Moreover, we used differential pulse voltammetry (DPV) for quantitative measurements. It was found that this new modified electrode linearly ranged from 0.001 to 700.0 μM, with detection limits of 0.34 nM and 0.22 nM for KYN and TRP, respectively. In addition, KYN and TRP in real samples can be analyzed by this sensor, with a recovery of 97.75%−103.6% for the spiked KYN and TRP in real samples.