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Zinc oxide (ZnO) nanoparticles (NPs) were fabricated by using leaves extracted from the thyme plant by employing a green method. The influence of several calcination (annealing) temperatures on the characteristic properties of fabricated ZnO NPs and the optimum calcination temperature for growing ZnO NPs were studied and reported. The studied calcination temperatures were 150 °C, 250 °C, 350 °C, and 450 °C. Different characterization techniques were used to study and examine the properties of biosynthesized ZnO NPs by using thyme plant leaf extract. The results of each UV-Vis analysis and FTIR spectrum of the leaf extract of the thyme plant confirmed and suggested that the selected leaf extract of thyme is a practicable choice for green synthesis of ZnO NPs. The investigated UV-Vis spectra of plant leaf extract displayed two strong absorption peaks at 266 nm and 313 nm at ambient temperature. The results of FESEM images showed that the calcination temperature has a significant and large effect on the morphology, size, shape, and orientation of ZnO NPs, which have a spherical shape with an average size in the range of 39.4–51.86 nm. In addition, the XRD results confirm that the ZnO NPs formed are pure ZnO with wurtzite hexagonal structure with particle size along the (002) peak in the range of 35.20–243.3 nm. The results of UV-Vis of ZnO NPs displayed a strong peak for all ZnO NPs produced at different calcination temperatures, a high absorbance in the UV region below 400 nm, and a low absorbance rate in the visible range. The obtained energy band gap (Eg) was in the range of 2.645–2.7 eV. In addition, the results of the FTIR spectra of ZnO NPs at different calcination temperatures revealed there was no discernible peak in the monitoring range, which indicated the purity of the ZnO nanoparticles generated via using thyme leaf extract. In addition, from all obtained results of the fabricated ZnO NPs, the ZnO NPs synthesized at the calcination temperature of 450 °C showed a high quality and improvement compared to the ZnO NPs synthesized at other calcination temperatures.