Premium ecosystem quality is an important guarantee for maintaining the supply and demand of human society and achieving sustainable development goals. Aimed at complex and diverse naturally geographical environment and obvious climate difference between regions in Nepal, the evaluation method based on reference conditions can obtain the relatively horizontal value of the ecosystem quality, which can reflect the different changing information of regions. Vegetation is an important indicator of changes in the quality of regional ecosystems. In view of the characteristics of large differences in altitude and climate conditions and rich vegetation types in Nepal, this study proposed a method of ecosystem quality evaluation based on the reference conditions and then analyzed its spatio-temporal pattern changes. First, twenty ecological assessment units were divided by five major geographical divisions and four main vegetation types in Nepal, meanwhile the relative vegetation index (RVI) was calculated from characteristics of three aspects of vegetation ecosystems:horizontal structure, production function and vertical structure. Then, a new vegetation ecosystem quality index (VEQI) was constructed by using the method of principal component analysis, and the ecosystem quality evaluation model based on reference conditions was constructed according to the reference areas from many national parks, wildlife reserves and nature reserves in Nepal. Finally, the vegetation ecosystem quality index based on reference conditions (VEQI') was calculated and it's spatio-temporal pattern changes was analyzed in 2016 and 2020. The results showed that (1) from 2016 to 2020, the average real value of Nepal's VEQI changed from 50.95 to 52.73, increased by 3.49%; the overall mean of VEQI' changed from 76.24 to 77.32, increased 1.42%; on the whole, the VEQI' improved in the context of improved reference ecosystem quality (VEQI_ref) (about 1.41%). (2) Using the 89% quantile of VEQI in ecological assessment areas as the reference values had a strong correlation with it's national protected areas. Consequently, we used them to replace as the reference values of the ecological assessment areas when the number of pixels in the reference area was small or the data quality was not good. (3) From the perspective of spatial change, the areas where ecosystem quality improved, basically stable and worsened in Nepal accounted for 74.16%, 14.25% and 11.59% of the total area of vegetation type ecosystems, respectively. Compared with data from field observation stations that are insufficient and difficult to collect and utilize, national nature reserves are closer to the assumption of an ideal reference ecosystem, rapid assessment of ecosystem quality can be achieved by determining the reference value of the ecological assessment area through limited nature reserves. This kind of evaluation method based on reference conditions can eliminate the differences in ecosystem quality caused by vegetation properties and natural geographical conditions and it's calculation results are more comparable in time and space, which can provide reference for regional ecological quality change assessment and quantitative analysis.