The uniformity and stability of bolt clamping force are critical factors influencing structural reliability. This study investigates the effects of different tightening sequences on bolt preload loss and the interaction between bolts during the tightening process. Through experimental testing, the variation patterns of bolt clamping force under three different tightening sequences were analyzed. Real-time monitoring data were used to evaluate the impact of tightening sequences on preload loss. Additionally, bolt relaxation behavior was observed over a 24-h period after tightening to assess long-term performance changes. The results indicate that tightening sequence significantly affects the clamping force, with each subsequently tightened bolt causing approximately a 1% loss in the clamping force of the previously tightened one. The natural relaxation behavior of bolts also varied under different tightening conditions, with preload loss within 10% over 24 h. Among the three tested sequences, the second sequence—in which bolts were tightened from the periphery toward the center—resulted in a clamping force loss of only 5.28% during the relaxation phase, suggesting it may be a superior tightening method compared to the one specified in standard procedures. This study provides experimental support for optimizing bolt preload design and offers theoretical insights into preload relaxation behavior, contributing to improved long-term stability of bolted connections.