A method using atomic force microscope (AFM) optical levers and a reference nanopositioning stage has been developed to characterize piezoscanner hysteresis and creep. The piezoscanner is fixed on a closed-loop nanopositioning stage, both of which have the same arrangement on each axis of the three spatial directions inside the AFM-based nanomanipulation system. In order to achieve characterization, the optical lever is used as a displacement sensor to measure the relative movement between the nanopositioning stage and the piezoscanner by lateral tracking a well-defined slope with the tapping mode of the AFM cantilever. This setup can be used to estimate a piezoscanner's voltage input with a reference displacement from the nanopositioning stage. The hysteresis and creep were accurately calibrated by the method presented, which use the current setup of the AFM-based nanomanipulation system without any modification or additional devices.