Abstract: The present disclosure relates to the technical field of biomedicine, and provides an anti-physaliatoxin single-domain antibody, and a preparation method and use thereof. The single-domain antibody is a VHH antibody with an amino acid sequence shown in SEQ ID NO. 1. Affinity analysis shows that the single-domain antibody of the present disclosure has prominent affinity. It is proved by small animal experiments that, after mice in an antibody protection group pre-injected with the single-domain antibody of the present disclosure are injected with physaliatoxin, no mice shows toxic symptoms, and during continuous observation for one month, no toxic lethality occurs, indicating that the single-domain antibody of the present disclosure shows excellent anti-physaliatoxin effects, excellent preventive or therapeutic effects on jellyfish stings, and promising clinical application prospects.