Vibration has an effect on structures. Vibration can affect structural stability by loosening the fasteners and might even cause structural failure. Experimental analysis on the pre-tightening force of the bolts in actual engineering was conducted to reveal the effect of the pre-tightening force of the fastening bolts on the dynamic behaviors of the structure. The bolt vibration isolation design was conducted using the force analysis of the fastening bolts, and the bolt vibration isolation measurement model was established. The expression of the maximal working interval of the pre-tightening force was deduced from experimental measurements of the spring washer, elastic vibration isolation content, and pretightening force. The compression test of the testing spring was used to determine the corresponding frequency, and a time history diagram was developed. The testing data were later analyzed to obtain an effective working interval of the system, an optimal pre-tightening force of the spring, and an effective stroke of displacement. Results indicate that different preload forces exert different vibration isolation effects on the structure. The spring stiffness is close to infinity when the spring is compressed to 24 mm, and the effective stroke is zero. The study provides guidelines for the design steps of the vibration isolation system based on its dynamic requirements and serves as a guide for selecting the appropriate spring washers and elastic vibration isolation materials in the project to achieve appropriate vibration isolation and noise reduction.