Abstract: Calcium ions play a central role in regulating plant growth, development and adaptation to environmental stresses. Calcium signals are one of the main regulators of plant growth, development and stress response, and calcium-binding proteins have also been identified as one of the most important components of calcium signal transduction pathways in plants. However, the assay techniques of calcium-binding proteins binding to calcium ions have not yet been evaluated in vitro and in vivo. Here, from the viewpoints of qualitative binding, quantitative binding and binding patterns, recent progresses in the principles, techniques, characteristics and applications of in vitro and in vivo calcium ion binding assays of calcium-binding proteins are reviewed, and the main detection methods and development trends in this field in recent years are also discussed in detail. Meanwhile,this review provides a new platform for isolation, functional identification and regulation mechanism studies of plant calcium-binding proteins in the future.