Remote sensing ecological index (RSEI) is widely utilized for ecological environment quality (EEQ) assessment. However, the consistency and difference in the application of RSEI constructed from Landsat and MODIS data in plateau regions have not been quantified. We conducted a comparison of RSEI_Landsat (RSEI derived from Landsat datasets) and RSEI_MODIS (RSEI derived from MODIS datasets) for EEQ assessment in the Yunnan-Guizhou Plateau (YGP). Then, the spatio-temporal dynamics and spatial heterogeneity of EEQ in the YGP from 2000 to 2022 were explored. The results indicated that: (1) There was a high correlation between RSEI_Landsat and RSEI_MODIS, with the R ranging from 0.83 to 0.89 in the YGP during 2000–2022. However, the RSEI_MODIS values were slightly lower than RSEI_Landsat in the transition years (2000, 2005, 2010, 2015, 2020, and 2022), with an average bias value of −0.06 and percent change (PC) value of −9.35%. (2) RSEI_Landsat exhibited an obvious advantage and sensitivity for evaluating EEQ and its dynamics compared to RSEI_MODIS in the YGP with high landscape fragmentation. (3) EEQ had a slightly improving trend, with average growth rates of 0.0054 a⁻¹ for RSEI_Landsat and 0.0037 a⁻¹ for RSEI_MODIS from 2000 to 2022 in the YGP. (4) Local spatial heterogeneity of EEQ revealed an increasing trend in the Yunnan-Guizhou Plateau. The study quantitatively evaluated the consistency and difference of the application of RSEI based on Landsat and MODIS datasets in plateau areas, which can provide a reference for the selection of data sources in regional EEQ evaluation.