It is critical to reliably and rapidly detect multiple disease biomarkers in tiny liquid samples with high sensitivity to meet the growing demand for point-of-care diagnostics. This paper reports a microfluidic platform integrating magnetic-based single bead trapping in conjunction with acoustic micromixing for simultaneous detection of multiple cancer biomarkers within minutes. Individual beads retained by permalloy (NiFe81/19) microarray were used to capture biomarkers and facilitate the fluorescence identification. A numerical study indicates that the magnetic force keeping a bead in the trap is proportional to the thickness of the permalloy array and the external magnetic field strength, while inversely proportional to the size of the trap. The acoustic microstreaming activated by a piezo transducer was applied to generate fast-switching flow patterns to minimize the diffusion length scales. The flow at various driving frequencies was experimentally tested to achieve the optimal mixing effect. The flow field of the microstreaming was subsequently described by a mathematical model to understand the flow further. Finally, the prostate-specific antigen (PSA) and carcinoembryonic antigen (CEA) were employed as model analytes to demonstrate the capability of the platform for rapid biomarker detection. With the aid of acoustic micromixing, the detection can be finished in 20 minutes. The respective limit of detection of PSA and CEA is 0.028 ng mL -1 (0.8 pM) and 3.1 ng mL -1 (17 pM), which is respectively 1/142 and 1/3 of the cutoff value of PSA and CEA. Our results indicate this platform has great potential for the rapid detection of multiple biomarkers in future point-of-care diagnostics.