Timely evaluation of habitat quality on national key ecological function zones for biodiversity conservation is an important scientific basis for the strategic planning and implementation measures of national biodiversity conservation. In this study, three factors including structure and function of ecosystem, ecological stress from social activities and response of human, were given priority consideration in the process of constructing a comprehensive index system of biodiversity assessment combining with previous research and pressure-state-response (PSR) framework model. Also, all indicators were processed by using weighted processing and normalization method and the comprehensive evaluation method of regional biodiversity were constructed based on remote sensing and GIS technology. Finally, the habitat quality status were assessed on the national key ecological function zones of biodiversity maintenance in 2011 and 2019. The results show that:(1) The population emigration generally occupy the dominant position in the national key ecological function zones of biodiversity maintenance from 2011 to 2019, but road net density has increased significantly. The human interference index and the fragmentation degree of ecological space increases accordingly. (2) The comprehensive index of biological diversity assessment (BDI) in all ecological function zones generally were maintained at above 0.4, and ecological environment quality status were in mid and good grade in 2011 and 2019. (3) The average BDI value of ecological function zones increased from 0.582 to 0.606, and the proportion of ecological function zones in the mid and good grade increased from 61.5% to 76.9% from 2011 to 2019. (4) The BDI value of two ecological function zones has decreased, occupied 15.4% from 2011 to 2019. In the last ten years, the habit qualities in the national key ecological function zones of biodiversity maintenance generally presents a trend toward better change. But controlling density of population, road network construction and ecological space fragmentation, and the construction of protection pattern based on the new territorial space planning, still are the key issues of biological diversity protection within a certain period. But population density, is critical pressures to biodiversity protection during future period of time, and increasing the area of nature reserve and strictly controlling is an effective pathway to relieve the stress. This evaluation method and index system had the characteristics of strong timeliness, simple operation and high efficiency, which could better reflect the actual status and change characteristics of biodiversity in the national key ecological function zones of biodiversity maintenance, and could be applied to regional scale biodiversity monitoring and evaluation.