Nickel resources are abundant in the world, and the application of nickel in production and life is more and more extensive. However, excessive nickel entering the environment will not only cause environmental pollution but also seriously endanger plants, animals and human health. Nickel compounds are carcinogenic and have been classified as a class 1 carcinogen. Nickel mainly exists in the form of divalent ions in the environment. However, there are few simple and effective methods for the detection of nickel ions, and these methods still have certain limitations. At present, the mechanisms of nickel influence in organisms are also unclear. Therefore, we constructed a ratiometric fluorescent probe Ra-Ni, which can achieve its own self-calibration and avoid the interference of other factors, thereby realizing the specific identification of nickel ions. The probe can detect nickel ions sensitively with a detection limit as low as 26.2 nM and can respond in a short time (< 2 min), which proves the great potential of the probe in the detection of nickel ions. At the same time, Ra-Ni has also been successfully used for imaging nickel ions in living cells and zebrafish, providing an effective tool for the study of physiological and pathological processes. The detection effect of nickel ions in actual water sample is also satisfactory, which further demonstrates the practicability of Ra-Ni in environmental applications.