Bolted joints are extensively utilized in aviation, aerospace, railway, and automotive engineering, where accurate control of preload is essential to ensure the structural integrity and operational safety of assemblies. To accurately measure the bolt preload, a bolt preload accurate measurement system was designed and developed based on the ultrasonic acoustoelastic effect, combined with the ultrasonic time-of-flight (TOF) accurate measurement technology. A refined theoretical model was first established to quantify the relationship between bolt preload and the variation in ultrasonic TOF. Subsequently, a high-voltage spike pulse excitation circuit was designed to effectively drive the ultrasonic transducer. A transmit/receive switching module and a cascaded amplification circuit were developed to obtain distinct echo signals. To achieve high-resolution temporal measurement, a method combining coarse and fine time conversion based on a time-todigital converter (TDC) chip was proposed. A dedicated hardware/software platform was implemented for ultrasonic TOF acquisition inside bolts. Finally, calibration experiments on M12 and M14 bolts were conducted using a universal testing machine, and a linear correlation between bolt preload and ultrasonic TOF variation was established. Experimental results validate that the proposed system achieves a preload measurement error of less than 5% when preload is greater than 15 kN. Moreover, the measurement error decreases as preload increases.