Abstract: The present invention belongs to the technical field of nanobodies, and more particularly, relates to an anti-ouabain nanobody and an application thereof. An amino acid sequence of the anti-ouabain nanobody disclosed by the present invention is as shown in SEQ ID NO. 1. A nucleotide sequence of the nanobody is shown in SEQ ID NO. 2. The nanobody has good affinity to ouabain, and can neutralize and antagonize the pharmacological activity of the ouabain, and detect the content of ouabain in a solution in combination with an ELISA method. The present invention provides a method for constructing the ouabain antibody with light weight, high thermal stability and high detection sensitivity; and provides a research tool for deeply excavating new biological functions of endogenous ouabain.