The 972 bp length of transaldolase gene tal was cloned from Pichia stipitis CICC1960, encoding a 323 amino acid protein with a calculated molecular mass of 35.36 kDa and isoelectric point of 5.20. Real time PCR analysis demonstrated that the mRNA transcript level of constitutive tal gene rise on xylose, glucose, fructose, mannose, galactose and sucrose as carbon source, respectively. Furthermore, the transcription of tal gene in P. stipitis on xylose was higher than on other carbon source, indicating that transaldolase plays a part in xylose utilization. To deeply study the tal gene biological function, it was expressed in Fusarium oxysporum CCTCC M209040. Recombinant transaldolase activity of transformant F. oxysporum M209040-Tal2 was about 0.52 U mg(-1) protein and was 1.57 times higher than that of the wild type F. oxysporum CCTCC M209040, indicating that the improvement of transaldolase activity in transformant was due to expression of the exogenous tal gene. Growth of transformant F. oxysporum M209040-Tal2 without selection pressure did not affect the level of hygromycin resistance of the transformants, suggesting that integrated tal gene was stable in mitosis. Fermentation trials of F. oxysporum M209040-Tal2 showed that the ethanol yield improved by 8.39 and 11.71% on glucose and xylose substrates, respectively, demonstrating that the expression of tal gene from P. stipitis CICC1960 in F. oxysporum CCTCC M209040 could improve ethanol production.