The invention discloses an in-service bolt axial force detection method, a detection system and a detection device. The detection method comprises steps: the bolt diameter of a bolt sample, a thread diameter, a sound velocity before force, a nut axial length, a screw rod length, a bolt force bearing area length, actually-measured axial forces of the bolt sample under different loads, ultrasonic transit time corresponding to the actually-measured axial force, and an ultrasonic transit time-axial force model are acquired; according to the bolt diameter, the thread diameter, the sound velocity before force, the nut axial length, the screw rod length, each actually-measured axial force, the ultrasonic transit time and the ultrasonic transit time-axial force model, a soft measurement expressionis determined; and by adopting the soft measurement expression, the axial force of the to-be-detected in-service bolt is calculated. The detection method provided in the invention not only takes intoaccount the influences of material constants of the bolt on the bolt axial force, but also takes into account influences of the length of the nut force bearing area and a nut force bearing area internal axis normal stress factor on the bolt axial force, the axial force of the in-service bolt can be effectively measured, the measurement errors are small, and the precision is high.