Stomatal closure, driven by shrinking guard cells in response to the accumulation of abscisic acid (ABA) under drought stress, has a great impact on plant growth and environmental acclimation. However, the molecular regulatory mechanism underlying the turgor alteration of guard cells remains elusive, especially in cereal grasses. Here, we develop a modified enzyme digestion-based approach for the isolation of wheat (Triticum aestivum L.) guard cells. With this approach, we can remove mesophyll, pavement cells and subsidiary cells successively from the epidermis of the trichomeless coleoptile in wheat and preserve guard cells on the cuticle layers in an intact and physiologically active conditions. Using a robust single-cell-type RNA sequencing analysis, we discovered 9829 differentially expressed genes (DEGs) as significantly up- or down-regulated in guard cells in response to ABA treatment. Transcriptome analysis revealed a large percent of DEGs encoding multiple phytohormone signalling pathways, transporters, calcium signalling components, protein kinases and other ABA signalling-related proteins, which are primarily involved in key signalling pathways in ABA-regulated stomatal control and stress response. Our findings provide valuable resource for investigating the transcriptional regulatory mechanism underlying wheat guard cells in response to ABA.