Gene expression is regulated by cis-regulatory elements. Assay for Transposase Accessible Chromatin sequencing (ATAC-seq) is an emerging technology that can accurately predict cis-regulatory elements in animals and plants. However, the presence of cell walls and chloroplasts in plants hinders the extraction of high-quality nuclei, thereby affecting ATAC-seq data quality. Meanwhile, it is tricky to extract high quality nuclei from various tissue types, especially for some tissue samples with limited size and amount. Moreover, with rapid growth of ATAC-seq datasets from plants, powerful and user friendly online pipelines for ATAC-seq data analysis is of particular interest. This study aims to establish an universal ATAC-seq experimental protocol suitable for multi-tissue types in wheat and to build ATAC-seq data analysis pipeline for plant. Using the newly development protocol, we efficiently obtained nuclei with less cell debris from various tissues of wheat and obtained high-quality ATAC-seq data from young spike and ovary, which is hard to harvest. We also investigated the saturation threshold of sequencing depth for wheat ATAC-seq, which turned out to be 10-20 G. In particular, we developed a user friendly online pipeline to analyze ATAC-seq data. The protocol and the analytical tool to map open chromatin here will facilitate plant regulatory genome study not only for wheat but also other plant species.