The invention, which belongs to the technical field of the high-speed main shaft on-line dynamic balancing, provides an adjusting method of a built-in mechanical on-line dynamic balancing system of a main shaft. The method comprises: step 1, installing an on-line dynamic balancing system; step 2, carrying out calibration of on-line dynamic balancing correction; to be specific, calibrating two adjustment balancing surface axial position parameters 1A and 1B; step 3, carrying out dynamic unbalance measurement and carrying out calculation to obtain a dynamic unbalance equivalent centrifugal force FD and location thereof 1D; step 4, carrying out dynamic unbalance correction of the main shaft; to be specific, carrying out calculation based on the obtained parameters to obtain an needed angle beta 1 or beta 2 for a mass block for adjustment, controlling the motor to make rotation, and adjusting the corresponding mass block; and step 5, displaying the dynamic unbalance data of the main shaft and adjustment data of the mass block on a display device of an industrial personal computer in real time to realize real-time monitoring of the whole process. According to the invention, the method is suitable for operation conditions of various main shafts to realize on-line correction of the dynamic unbalance states of the main shafts. The rotation precision of the main shaft is improved, thereby realizing processing of high-quality parts.