Abstract: Disclosed are the expression of a recombinant chicken IgY scFv antibody and monoclonal antibody raised against human thymidine kinase 1 (hTK1) in prokaryotic cells and a preparation method and use thereof. The scFv antibody includes an amino acid sequence shown in SEQ ID NO: 1. The anti-hTK1 recombinant scFv antibody and monoclonal antibody have good immunoreactivity, high specificity and sensitivity, and are easy to separate and purify.