Key Biodiversity Areas (KBAs) are ecological conservation priorities proposed by IUCN and widely recognized by most countries. Evaluating the changes in the ecological characteristics in KBAs is important for biodiversity conservation and the construction of Protected Areas (PAs). There are various ecosystem types in the Belt and Road Initiative (BRI) region, which has an extremely high value of biodiversity conservation, and the KBAs should be the prime targets of ecological protection efforts. Using the data of land cover, NDVI and Nighttime Light (NTL), we analyzed the ecological conditions of the KBAs in the BRI region, and their temporal and spatial variations, from the perspectives of vegetation coverage and human activities. The conclusions are: (1) There is generally no significant difference in the land cover of the KBAs, among which forest, wilderness and grassland are the main types; (2) The NDVI of the KBAs showed an increase, indicating that the vegetation was gradually improving, while a few KBAs presenting vegetation degradation were mainly distributed in the Indochina Peninsula, Qinghai-Tibet Plateau and Central and Western Asia; and (3) The NTL in the KBAs was very low, indicating that the human pressure on the natural ecosystems was limited, and only a few KBAs distributed in Central and Eastern Europe, India, and the Indochina Peninsula have high human activity intensity which also showed an increase. This study emphasizes that we should make full use of the biome succession law, and limit the interference of human activities on natural ecosystems for ecological protection of the KBAs, so as to continuously make new breakthroughs in the construction of Protected Areas (PA) in the BRI region.