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A true non-intrusive 3D Micro-Acoustic Trap for a controlled monolayer assembly of microparticles in aqueous environment. Modulation of acoustic pressure generated by an acoustic lens can assist in particle manipulation and formation of a monolayer of polystyrene beads suspended in aqueous media. The same experimental model can also be used for manipulating and probing live cells. • Micro-Acoustic Trap for controlled assembly of microparticles as a monolayer suspended in aqueous media. • A suspended 2D monolayer of polystyrene beads with hexagonal close pack lattice with high density of particles. • Multilayered monolayer arrangement of microparticles in 3D. • Acoustically controlled mobility and segregation of live cell. Acoustic tweezers facilitate the manipulation of objects using sound waves. With the current state of the technology one can only control mobility for a single or few microparticles. This article presents a state of the art system where an Acoustic Lens was used for developing a Micro-Acoustic Trap for microparticle assembly in 3D. The model particles, 2 µm diameter polystyrene beads in suspension, were driven via acoustic pressure to form a monolayer at wavelength-defined distances above the substrate defined by the focal point of an Acoustic Lens The transducer was driven at 89 MHz, mixed with 100 ms pulses at a repetition rate of 2 Hz. Beyond a threshold drive amplitude sufficient to overcome Brownian motion, this led to 2D assembly of the microparticles into close-packed rafts >80 µm across (∼5 wavelengths of the carrier wave and >40 particles across). This methodology was further extended to manipulation of live Dictyostelium discoideum amoebae. This approach therefore offers maneuverability in controlling or assembling micrometer-scale objects using continuous or pulsed focused acoustic radiation pressure. [ABSTRACT FROM AUTHOR]