Desert areas occupy approximately 25% of total land area and are characterized by scarce precipitation, poor soil conditions, and a harsh ecological environment. Desertification may result in the loss of unique biological resources. Therefore, exploring the biodiversity of deserts and the causal factors of desertification is necessary to develop future solutions to global desertification. The desert area in northwest China is an important part of the arid zone of central Asia as it was traveled during the ancient Silk Road and witnessed the rise and fall of many civilizations, making it an ideal location for desertification research. This study utilized environmental DNA in the surface soil along the eastern part of the Silk Road and DNA metabarcoding technology. Bioinformatic analysis identified 782 plant species belonging to 505 genera and 133 families, and plant species diversity gradually decreased from east to west along the Silk Road. Temperature, altitude, and longitude were the main factors affecting plant species diversity in the area, while the impact of precipitation was relatively small. Correlation analysis between species diversity, population density, and major human settlements revealed that human activity significantly affected plant species diversity. Our study successfully used environmental DNA and metabarcoding technology to reveal the plant species diversity and its influencing factors for a large-scale desert area, which will provide a fundamental and theoretical basis for desert management and biodiversity protection.