Aiming at the on-line calibration problem of in-service strain monitoring systems ( SMS) for bridge structures under all-day working conditions, an on-line calibration method based on passive excitation is proposed, and an on-line calibration system model is established. In this model, the dynamic load of normal passing vehicles on the bridge is used as the excitation source. Through synchronously measuring the structural strain response parameters of the SMS and the reference system, the traceability chain of calibration is constructed to realize the on-line calibration under the continuous working state of the in-service SMS. According to the requirements of the measurement performance evaluation of the SMS, a quantitative analysis model of period measurement error, basic error and confidence interval based on large sample data is established. Experiment verification was carried out on Jiujiang Bridge in Guangdong Province. The results show that the proposed method has feasibility for field implementation, and the on-line calibration results calculated from different data sets have good consistency. When the coverage probability is greater than 90% , the half-width deviation of the basic error interval is less than ±0. 005.