The production of cellulase from Penicillium expansum MDFS2 on mixed substrates of barley straw and wheat straw (1:1) was investigated. The highest exoglucanase (6.72 U/g) and endoglucanase (40.75 U/g) activities observed on the 5th day of fermentation process. However, the highest β-glucosidase activity (15.47 U/g) was on the 6th day of fermentation process. The optimum temperature for exoglucanase and β-glucosidase activity recorded at 40°C and β-glucosidase retained more than 70% of its maximum enzymatic activity at 80°C. Also, the highest endoglucanase activity was recorded at 50°C. The highest activity for all the three major components of cellulase enzyme complex was observed at pH= 5.0. The enzyme activity reduced with increasing in temperature and incubation period. The results showed that β-glucosidase is more stable at 50°C in comparison of endoglucanase and exoglucanase and also 80% of β-glucosidase original activity was maintained 3-h after incubation period. According to the results of this study, Penicillium expansum MDFS2 could be suggested as a strain with good potential for the growth on the mixed substrates of barley straw and wheat straw for cellulase production.