Green roofs provide ecosystem services and can promote biodiversity in urban areas. Blue-green roofs have an additional water storage compartment under the substrate to reduce roof water runoff, thereby also reducing drought stress which is beneficial for green roof vegetation. In order to study which blue-green roof design supports the highest plant diversity, we assessed the effect of different substrates and seed mixtures on vegetation development in a short-term greenhouse experiment and long-term blue-green roof experiment. A ten-week full-factorial greenhouse experiment was performed for six substrate composition and four seed mixture treatments. On an experimental blue-green roof, we annually surveyed plants from 2013 to 2021 in nine different treatments (five replicates each), that varied in substrate composition, substrate depth and seed mixture that was initially applied. Two treatments resembled conventional non-green roofs (100% gravel) and conventional extensive Sedum green roofs. The results of the greenhouse experiment showed that seed mixture is more important than substrate composition in shaping the initial species richness and species composition. However, on the experimental roof the substrate composition was an important determinant of species richness and species composition long-term. Plant species richness on the experimental roof was lowest in the gravel treatment (resembling conventional non-green roofs), and highest in treatments where locally collected soil was used, likely due to additional species that appeared from the seed bank present in the transplanted soil. Soil was never completely covered with vegetation on unfertilized substrates that contained 20% or less dense and organic materials. Plant species richness on conventional Sedum roof substrate was higher on the experimental blue-green roof compared to an adjacent non-blue roof, highlighting that blue-green roofs can promote biodiversity more than conventional green roofs. For future construction of blue-green roofs in our region, we recommend the addition of 30% locally collected soil to a 6 cm deep lightweight substrate to maximize long-term plant cover and plant species richness.