The bead-based immunoassay requires not only efficient mixing but also good control of bead-surface-area-to-sample-volume ratio to realise accurate and reproducible detection of low concentration samples. This paper reports the development of a microfluidic platform with the reproducible and efficient bubble-induced micromixing for bead disaggregation and immunoassay of prostate-specific antigen (PSA). The platform consists of a microfluidic chip with a microchamber and rectangular traps for capturing air bubbles and a home-made controller to generate sound wave using an external piezo transducer. Methods for reproducible bubble formation and bubble size control during mixing are explored. The influence of driving voltage, PDMS thickness and the substrate material on the mixing efficiency is characterised by mixing a fluorescence dye and a buffer solution. The optimised acoustic microstreaming is able to break clusters with hundreds of beads and homogenise individual beads over the microchamber. Immunoassay with efficient micromixing has been applied to PSA immunoassay with greatly reduced detection time. This study provides a practical guide for the design and development of the bubble-induced acoustic micromixers for bead disaggregation and on-chip immunoassays.