Based on a β-tubulin gene EST sequence from Trichoderma harzianum mycelium cDNA library, a 1.74kb β-tubulin gene coding region, together with a 1.5kb 5′-flanking region and a 1.0kb 3′-flanking region was cloned from T. harzianum by IPCR methods. The gene encodes a 446 amino acid polypeptide, which shows a high degree of homology with other fungal β-tubulins. A three-dimensional model of the T. harzianum β-tubulin was built based on the crystal structure of bovine tubulin using homology modeling method. Tertiary protein structure of the β-tubulin should provide a basis for understanding a significant body of research on tubulin′s properties and interactions with antimicrotubule agents.