The physiological maturity of cow (cow age) is considered to be an important indicator of beef quality classification. For development of beef quality grading standard and the automatic beef grading system, a quantitative relationship between the age and tenderness of beef plays an vital role which has to be established. To research the relationships among cow age, features of beef muscle fiber and beef tenderness, and four different groups of longissimus dorsi beef with different ages were selected, after been sliced up and stained, the microscopic images of the ribeye regions sample were collected by microscopic machine. Then the diameter, circumference and density of each muscle fiber were measured by using image processing. After determining the shear force of each beef sample by using a texture analyzer, the change of diameter, perimeter and density of beef fiber with age were analyzed，the relationship between the diameter, perimeter as well as density and the shear force were then established, respectively. The experiment result revealed that the diameter and circumference of beef fiber were increased，while the muscle fiber density was linearly decreased with the increase of age of the cow (P<0.05). At the same time, it was also observed that the shear force of beef was significantly increased with the increase of diameter and perimeter of muscle fiber and the decrease of muscle fiber density (P<0.05). It was concluded that the age of cattle had significant influence on the diameter, perimeter density of muscle fiber and shear force of beef. There was a significant correlation between the age of cow, the microparameters of beef muscle fiber and tenderness of beef, the relationship between them can be described with a linear model clearly.