The invention provides a pyridine-coumarin derivative fluorescent probe, and a preparation method and application thereof. The chemical structural formula of a pyridine-coumarin derivative is as described in the specification. The preparation method comprises the following steps: dissolving 7-hydroxy-3-(3-methylisoxadiazol-5-yl)coumarin and triethylamine in an organic solvent, dissolving pyridyl-2-formyl chloride in another organic solvent, and dropwise adding the dissolved pyridyl-2-formyl chloride into the formed former solution at a temperature of 0 DEG C; naturally heating a reaction mixture to room temperature, and performing continuous stirring for 3 h; and carrying out reduced-pressure suction filtration, subjecting a filtrate to reduced-pressure evaporation to dryness, and carryingout recrystallizing with ethanol to obtain the pyridine-coumarin derivative fluorescent probe. The pyridine-coumarin derivative fluorescent probe can selectively react with copper ions under physiological conditions, remarkably enhance the blue fluorescence of a solution, and is particularly applied to convenient detection of copper ions in cells as a fluorescent probe.