Diagnosis of the bolt loosening is important in the industrial field and the medical field such as dental implants. This study proposes a new non-destructive testing method for the bolt loosening using ultrasonic pulse projected to the side of the bolt. This method focuses on the reflected pulse from the screw thread and its instantaneous frequency. A basic experiment with aluminum block and a stainless steel bolt shows that the tightening torque of the bolt can be evaluated via the instantaneous frequency of the reflected pulse from the screw thread. Decreasing the tightening torque, the instantaneous frequency reach a maximal value earlier. We tried to verify the mechanism of the proposed method through an identification of the transmission paths and a numerical simulation. Real contact area on the load flank decreases, and the reflectance on the load flank changes when the tightening torque decreases. The analytical study shows that the change of the reflectance affects the received pulse. Especially, a peak time of the instantaneous frequency of the received pulse varies owing to the amplitude ratio of the reflected pulses from the load flank to the pulses reflected from the other contact surfaces. This analytical results follows the experimental results. Therefore, observing the peak time of the instantaneous frequency of the received ultrasonic pulse, we can obtain the reasonable index to diagnose the bolt loosening.