The invention relates to four green fluorescent protein (GFP) nano antibody encoding genes as well as a preparation method and application thereof. The invention establishes a GFP nano antibody library. By using a phage display technique, four nano antibodies which are specifically combined with the GFP are screened from the antibody library, which are respectively named as A12, E6, D5 and B9. Nucleotide sequences of the four nano antibodies are obtained through sequencing, the nucleotide sequences are as shown in SEQ ID NO:1. SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, and the nucleotide sequences have corresponding amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. A gene A12 is cloned to a transformed expression vector pADL-10b-His, and introduced intoan SS320 strain; genes E6, D5 and B9 are respectively cloned to transformed expression vectors pBAD 24-Flag-His, and are respectively introduced into a TOP10 strain, and then four prokaryotic expression vectors and strains of four nano antibodies are obtained. Four nano antibodies are expressed and purified, tests show that the four GFP nano antibodies can be specifically combined with GFP, and the genes can be applied to detection on GFP in basic research.