In the context of ZnO nanorods (NRs) grown on Si and indium tin oxide (ITO) substrates, this study aimed to compare their degradation effect on methylene blue (MB) at different concentrations. The synthesis process was carried out at a temperature of 100 °C for 3 h. After the synthesis of ZnO NRs, their crystallization was analyzed using X-ray diffraction (XRD) patterns. The XRD patterns and top-view SEM observations demonstrate variations in synthesized ZnO NRs when different substrates were used. Furthermore, cross-sectional observations reveal that ZnO NRs synthesized on an ITO substrate exhibited a slower growth rate compared to those synthesized on a Si substrate. The as-grown ZnO NRs synthesized on the Si and ITO substrates exhibited average diameters of 110 ± 40 nm and 120 ± 32 nm and average lengths of 1210 ± 55 nm and 960 ± 58 nm, respectively. The reasons behind this discrepancy are investigated and discussed. Finally, synthesized ZnO NRs on both substrates were utilized to assess their degradation effect on methylene blue (MB). Photoluminescence spectra and X-ray photoelectron spectroscopy were employed to analyze the quantities of various defects of synthesized ZnO NRs. The effect of MB degradation after 325 nm UV irradiation for different durations can be evaluated using the Beer–Lambert law, specifically by analyzing the 665 nm peak in the transmittance spectrum of MB solutions with different concentrations. Our findings reveal that ZnO NRs synthesized on an ITO substrate exhibited a higher degradation effect on MB, with a rate of 59.5%, compared to NRs synthesized on a Si substrate, which had a rate of 73.7%. The reasons behind this outcome, elucidating the factors contributing to the enhanced degradation effect are discussed and proposed.