We developed a human intracellular antibody based on the M1 protein from avian influenza virus H5N1 (A/meerkat/Shanghai/SH-1/2012) and then characterized the properties of this antibody. The M1 protein sequence was amplified by RT-PCR using the cDNA of the H5N1 virus as a template, expressed in bacterial expression system BL21 (DE3) and purified. A human strain, high affinity, and single chain antibody (HuScFv) against M1 protein was obtained by phage antibody library screening using M1 as an antigen. A recombinant TAT-HuScFv protein was expressed by fusion with the TAT protein transduction domain (PTD) gene of HIV to prepare a human intracellular antibody against avian influenza virus. Further analysis demonstrated that TAT-HuScFv could inhibit the hemagglutination activity of the 300 TCID50 H1N1 virus, thus providing preliminary validation of the universality of the antibody. After two rounds of M1 protein decomposition, the TAT-HuScFv antigen binding site was identified as Alanine (A) at position 239. Collectively, our data describe a recombinant antibody with high binding activity against the conserved sequences of avian influenza viruses. This intracellular recombinant antibody blocked the M1 protein that infected intracellular viruses, thus inhibiting the replication and reproduction of H5N1 viruses.