An asymmetric salamo‐based probe molecule (H2L) was synthesized and characterized structurally. When DMF/H2O (9:1) was used as the solvent, it was shown probe H2L has high sensitivity to Cu2+. Using high‐resolution mass spectrometry and theoretical calculation, it was found that probe H2L could form a more stable complex (1:1) with Cu2+, the minimum limit of detection (LOD) of H2L for Cu2+ was calculated as 9.95 × 10−8 M. In addition, probe H2L could also be used to identify B4O72− under the same detection conditions and the minimum LOD of H2L for B4O72− was calculated as 4.98 × 10−7 M. At the same time, density functional theory theoretical calculation further proved the flexibility of probe H2L. Through the action of EDTA, probe H2L had a cyclic ability to recognize Cu2+, and showed a better response in the physiological pH range; probe H2L had the characteristics of fast recognition speed and high efficiency. In addition, with probe H2L test paper for Cu2+ and B4O72−, the effect was more obvious. Meanwhile, probe H2L can be used to quantitatively detect Cu2+ in water samples.