Physical properties of materials such as tensile strength, toughness and hardness are usually measured by using destructive methods. The destructive evaluations are costly in many cases. Therefore, the use of non-destructive methods for evaluating the physical properties of materials, especially in production lines, is logical. Some physical properties, such as Young's modulus, etc., can be directly measured using ultrasonic waves based on the measurement of the velocity longitudinal and transverse waves. However, it is not possible to directly measure hardness with ultrasonic waves because the hardness depends on the parameters of microstructure of the material such as grain size, phase type in the material, phase size and other parameters. In this research, an attempt has been made to investigate the relationship between the attenuation of ultrasound and hardness of steel. The effect of different temperatures and heat treatment on microstructure and ultimately of ultrasonic wave’s attenuation have also been studied. . For this purpose, the C316 stainless steel which is widely used in marine, military, and chemical industries is selected for experiments. The specimen are under annealed heat treatment to create different microstructures by changing the temperature and time of the annealed operation. The specimen are then evaluated by ultrasonic waves and hardness test. Keywords : Ultrasound, hardness, ultrasonic waves, microstructure