［Objective］To exploring the spatio-temporal changes of long time series in habitat quality of the Wujiang River Basin in Guizhou Province，in order to provide a theoretical reference for high-quality green development of the region.［Methods］Based on four periods of land use data from 1990，2000，2010 and 2020， the landscape pattern and habitat quality of the Wujiang River basin in Guizhou Province were calculated and the driving factors were analyzed，by using the moving window method，InVEST model，and geographic detector. ［Results］1）From 1990 to 2020，the fragmentation and patch shape complexity of the landscape pattern increased within 30 km of the main stream in the south of the upstream，while the patch dominance and landscape connectivity decreased. The fragmentation and patch shape complexity of the landscape decreased at a distance of 30-60 km from the main stream in the south of the midstream，while the landscape connectivity increased. 2）The annual mean value of habitat quality was 0.67，with an increase of 1.67% in high habitats，0.17% and 1.46% in medium low and low habitats，respectively. The proportion of high habitats in the upstream and medium low and low habitats in the midstream increased the most. The proportion of high habitats at the distance of less than 30 km from the main stream and medium low and low habitats at the distance of 30-60 km increased the most. 3）Land use intensity was the main driving factor for spatial heterogeneity of habitat quality，while temperature and rainfall were secondary factors.［Conclusion］The overall habitat quality of Wujiang River basin in Guizhou Province is medium high，and the changes in some areas are due to the trade-off between ecological protection and economic development.