Habitat quality is an important index to estimate the biodiversity status and local ecological functions. Exploring the spatio-temporal evolution of regional habitat quality is of great importance in the context of high-quality development. Taking Zhejiang as the case study, we investigated the spatio-temporal evolution and influencing factors of habitat quality based on the InVEST model, the hot spot analysis, and the Geodetector. Meanwhile, we computed the habitat quality within the ecological red line areas in Zhejiang using the above assessment results. The results show that: (1) from 2000 to 2015, the mean value of habitat quality in Zhejiang showed a decelerating downward trend, and spatially formed a distribution pattern of high in the northwest, the southwest, the middle east, but low in the northeast and the middle; the habitat degradation presented a 'center-periphery' sphere radiation structure. (2) Based on the hotspot analysis, the agglomeration characteristics of habitat quality and habitat degradation were similar on the township scale, while the spatial distribution trends of cold and hot spots were opposite. (3) Based on the Geodetector, terrain factors (elevation and slope) showed the most significant impact on habitat quality, and the contribution to habitat quality from vegetation factors (NPP, NDVI) increased gradually with time. Meanwhile, the synergy of natural and socio-economic factors affected the spatial variation of habitat quality in Zhejiang. (4) The habitat quality of the ecological red line area in Zhejiang was relatively high and stable, and with differences among different types of ecological red lines. The areas with high habitat quality but not designated as ecological red line areas were mainly distributed in Zhejiang's southwest and northeast mountain areas, while less were found in the northern, central, and eastern Zhejiang. Therefore, we proposed complementary strategies to adjust the ecological red line areas, delimit the regional ecological function zonings, and finally improve ecological spatial management and control in Zhejiang.