Two-dimensional (2D) materials have many remarkable properties such as high conductivity, optical transparency and mechanical strength, their piezoelectric and piezoelectric photoelectron effects have attracted more attention, and the study of piezoelectric properties of two-dimensional materials is of great significance for the application of microelectromechanical systems, nanorobots, and active flexible electronic products. In this paper, atomic force microscopy (AFM) was used to investigate the anomalous off-plane piezoelectric effect of two-dimensional layered zinc oxide (ZnO) materials. First, ZnO single crystal microwires were synthesized by chemical vapor deposition, and then they were exfoliated by ultrasonic exfoliation to obtain two-dimensional layered ZnO nanosheets. Subsequently, the microstructure, crystal structure and growth orientation of ZnO single crystal microwires were characterized by scanning electron microscopy, transmission electron microscopy and EBSD. Then, the amplitude-bias voltage butterfly-like curve and the piezoelectric coefficient-bias voltage hysteresis curve were measured by the PFM module of AFM for two-dimensional layered ZnO nanosheets. The results show that ZnO single crystal microwires have a single-crystal wurtzite structure grown along the thickness orientation ［0001］. Two-dimensional layered ZnO nanosheets are of less than 10 nm thick, also have a single-crystal wurtzite structure grown along the thickness orientation ［0001］, and the effective out-of-plane piezoelectric coefficient can reach a maximum of 48.4 pm/V.