The invention relates to a tissue sealant and a preparation method and application thereof. The sealant mainly comprises hydrophilic raw material, nucleophilic raw material, a marker and physiological thinner, wherein the hydrophilic raw material mainly refers to the derivatives of polyethylene glycol and the nucleophilic raw material mainly refers to polyamino acid. The preparation method includes: physically mixing the hydrophilic raw material, the nucleophilic raw material, the marker and the physiological thinner through a mixing tool, and performing covalent cross-linking to form hydrogel. The tissue sealant has the advantages that the sealant mainly used for sealing small wounds after eye surgery is good in tissue cohesive force and film-forming property, good in biocompatibility, capable of promoting epithelization healing and preventing seepage leakage, simple in preparation method, capable of being quantified to form a stable production line, and good in clinical application value.