The invention discloses a relay-type multifunctional fluorescent probe, and a preparation method and application thereof, belonging to the field of functional materials. The preparation method comprises the following steps: adding N-[2-(5-phenyl-1,3,4-oxadiazolyl-2-yl)phenyl]-2-chloroacetamide, di-(pyridylmethylene)amine, N,N-diisopropylethylamine and potassium iodide into acetonitrile, and stirring and heating under reflux in a nitrogen protective atmosphere; and after the reaction finishes, distilling under reduced pressure to remove the solvent, carrying out column chromatography separation on the residues to obtain the fluorescent probe N-[2-(5-phenyl-1,3,4-oxadiazolyl-2-yl)phenyl]-2-di(2-pyridylmethyleneamino)acetamide. The fluorescent probe has excellent water solubility, can be used for monitoring analysis and tracing of zinc ions, pyrophosphate and sulfur ions in a water environment system, and has favorable determination sensitivity for target ions.