Abstract: Bulk acoustic wave (BAW), as a typical acoustic field manipulation method, enables direct alignment of microparticles in three-dimensional directions with excellent biocompatibility, and shows a broad application prospect in patterning arrangement of tiny biological targets. For tissue engineering, bio-3D printing and other related applications, the efficiency enhancement of microparticle patterning arrangement is a key factor for the final quality. Although theoretical descriptions of particle motion during the patterning process are available, the factors affecting the patterning efficiency are still unclear due to the inability to implement high-precision measurements at the microscale and the difficulty of constructing accurate and reliable simulation models. In order to investigate the main factors affecting the patterning efficiency and their characteristics, a set of visualization experimental platform was designed and constructed, and the motion behaviours of microparticles under the action of BAW and the state changes of individual microparticles were precisely observed by using a microscopic vision system, and the effects of the piezoelectric ceramic transducer driving voltage, suspension concentration, particle size, and liquid viscosity on the patterning time were analysed. This integrated approach not only advances our understanding of the underlying mechanisms governing microparticle patterning but also provides valuable insights for optimizing patterning processes in diverse applications. The results show that, in a specific range, the increase of microsphere suspension concentration leads to a slight prolongation of the patterning time, however, this difference is not significant; for microspheres with diameters between 10 μm and 100 μm, the increase of their diameters contributes to the enhancement of the patterning efficiency; and the liquid viscosity has the most significant effect on the patterning efficiency compared to other parameters. This study provides important data and design guidance for the study of BAW-based realisation of microparticle patterning and related applications, which can help to promote the development of biomedical, materials science and other related fields.