Triethylene-tetramine(TTA) and glutaraldehyde(GLU) were used to stabilize immobilized yeast particles. The orthogonal experiments were carried out to determine the effects of concentrations and treatment time of TTA and GLU on chemical stabilization of the immobilized yeast. The ethanol fermentation experiments were conducted in a 5 L bioreactor to investigate the influence of the stabilized immobilized yeast on ethanol fermentation of sweet sorghum stalk juice. The results showed that the optimum treatment for chemical stabilization of immobilized yeast particles was as follows: TTA concentration of 0.5%, TTA treatment time of 120 min, GLU concentration of 0.5% and GLU treatment time of 8 min. Eight batches ethanol fermentation results of sweet sorghum stalk juice in the 5 L bioreactor showed that the average yield of ethanol and coefficient of variation(CV%) were 84.78% and 8.08%, respectively, when the immobilized yeast particles stabilized by the optimum treatment were used for fermentation, while those of blank treatment were 84.32% and 9.68%, respectively. Therefore, the behavior of the stabilized immobilized yeast was slightly better than that of blank treatment. The research has provided a scientific reference for the researches of refining bioethanol from sweet sorghum stalk juice by immobilized yeast.