In the past two decades, the agro-pastoral ecotone of northern China has experienced dramatic habitat loss and fragmentation, and patches of remaining grassland have formed. However, it is still unclear how to prioritize biodiversity conservation in the remaining grassland patches in this fragmented landscape. Here, we investigated 84 remaining grassland patches in the Tabu River Basin, a typical area of the agro-pastoral ecotone of northern China. To determine the conservation priorities of these remaining grassland patches, we used a new conservation criterion called ecological uniqueness based on beta diversity. We further explored the impact of local habitat, dispersal process, and patch characteristics on ecological uniqueness. Our results showed that turnover was the dominant component of regional beta diversity (accounting for 81.9%). We identified 13 hotspots patches with high conservation priority due to their significant ecological uniqueness, 9 of which were small patches (<5 ha). Local habitat explained more variation of the ecological uniqueness than dispersal process and patch characteristics, with local precipitation heterogeneity the key influencing factor. We also found a positive correlation between patch fractal dimension and ecological uniqueness. Our study suggests that small remaining grassland patches are hotspots of regional biodiversity conservation in the fragmented landscape of the agro-pastoral ecotone of northern China. We highlight that it is necessary to focus on the habitat heterogeneity and patch shape of these small patches to determine conservation prioritization.