Changes in land cover and vegetation driven by climate change and human activities can both affect habitat quality. Qinghai-Tibet Plateau has very high biodiversity and provides a habitat for numerous wildlife species. The Lhasa River Basin, located in the southern part of the Tibetan Plateau, is where human activities are very intense, increasing the threat and pressure on habitat quality. To analyze the effects of land cover and vegetation changes on the habitat quality of Lhasa River Basin, we selected growing season (from June to October) Normalized Difference Vegetation Index (NDVI) as the index for vegetation condition to adjust the suitability index of different vegetation types and then assessed habitat quality change in Lhasa River Basin from 1990 to 2015 by using the InVEST model. Results showed that the overall change in land cover in Lhasa River Basin was relatively small, except for the sharp increase in artificial surface and wetland (increased by 82.65% and 32.40%, respectively). The main land-cover transfer directions were from sparse grassland to meadow and steppe, farmland to artificial surface, and ice/snow to barren land. Except for the barren land in the upper and middle regions of the basin as well as Nyenchen Tanglha Mountain, overall NDVI showed a significant increase from 1990 to 2000, and then a slight decrease thereafter. From the perspective of spatial distribution, the high habitat quality areas were distributed throughout the valley of the Nyenchen Tanglha Mountain as well as downstream and headstream of Lhasa River. The low habitat quality areas were mainly located at the construction sites of Lhasa City and Linzhou County and their surrounding areas, along with the barren land located in the upper and middle regions of the basin. From the perspective of temporal changes, the average habitat quality index increased significantly from 0.51 in 1990 to 0.57 in 2000; the average habitat quality indices of 2010 and 2015 were 0.56 and 0.55, respectively, and they showed a slight decrease when compared with the value in 2000. When compared with land cover change, the NDVI change had more significant effects on habitat quality in the study region.