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Structural anisotropy, often observed in naturally occurring materials such as wood and human tissues, is central to the function in several engineered and non-engineered applications. In this study, we present the theory and proof-of-concept demonstration of an ultrasound-assisted non-contact manufacturing approach to create well-defined spatial patterns of micro-particles within a fluid matrix. A chamber with opposing pair of ultrasonic transducers was designed and prototyped based on standing bulk acoustic wave theory, and it was used to study the effects of ultrasound frequency (1, 1.5, 2, 3 MHz) and voltage amplitude (80, 160 mVpp) on alignment characteristics of polymer micro-particles (mean Ø = 8 μm) suspended in water (0.01 g/ml). The experimental results were consistent with theory in that the micro-particles aligned along linear strands, with the inter-strand spacing reducing with increasing frequency (p < 0.05). Increasing voltage amplitude reduced the time taken to align the particles, but it did not significantly change the observed spacing (p > 0.05). The observed spacing, however, was higher than the theoretical spacing of half-wavelength, for each frequency and amplitude. The alignment of living human adipose derived stem cells in viscous alginate hydrogel matrix was also successfully demonstrated. The approach presented herein can be scaled up into manufacturing processes, including layered manufacturing, to create highly functional mechanically and/or electrically anisotropic composites through controlled spatial geometry, as well as to biofabricate engineered tissues with aligned cells and extracellular matrix components to mimic natural tissues.