The invention discloses a positioning and assembling structure of a force measurement unit and a use method, and belongs to the field of piezoelectric force measurement. Two sliding grooves are formed in a middle convex surface of a convex bottom plate and connected with a sliding plate through bolts. A lateral plate and a rear plate are connected with the convex bottom plate through two rows of bolts arranged side by side. The L-shaped sliding plate, the lateral plate and the rear plate are each provided with two sliding grooves which are connected with sliding rails through bolts. The sliding plate and the rear plate are each provided with two sliding rails. The lateral plate is provided with one sliding rail. Each sliding rail is provided with two sliding blocks. The sliding blocks and the sliding plate can be adjusted at multiple degrees of freedom, accurate positioning is achieved between a test piece and the structure, assembling and application of pre-tightening force are carried out, and assembling of force measurement units of different size models and application of the pre-tightening force are realized by means of base blocks of different models. The problem that when the pre-tightening force is applied to the force measurement unit, as friction force exists between an upper pressing block and a pre-tightening nut, the upper pressing block and a lower pressing block can not be aligned is solved, the assembling efficiency and assembling accuracy of the force measurement unit are improved, the labor intensity is relieved, the structure is simple, and the use is easy and convenient.