Quantitation of protein biomarker featured with portability, rapidity, high sensitivity is critical for the point-of-care testing (POCT) application. Herein, a novel smartphone-enabled microfluidic chemiluminescence platform for the quantitation of prostate specific antigen (PSA) was proposed based on acoustic tweezers approach. The primary antibodies labeled polystyrene microparticles (Ab1-PSs), target samples, the horseradish peroxidase labeled secondary antibodies (Ab2-HRP) were injected into the microfluidics simultaneously. Under the actuation of Lamb Wave Resonator (LWR), they were dynamically trapped and concentrated in the acoustic streaming; meanwhile, the biomolecular binding was enhanced. After the injection of chemiluminescent substrate, the concentrated immuno-particles catalyzed hydrogen peroxide (H2O2) reaction so that the emitted blue light was directly captured by smartphone. Besides, the flow rate and the applied power of LWR were optimized for the signal amplification. The chemiluminescence immunoassay exhibited a dynamic linear range from 0.5 ng/mL to 10 ng/mL with a limit of detection of 0.1 ng/mL in PBS buffer. The portable immunosensor will be utilized for the quantitation of PSA in serum samples to demonstrate the clinical significance.Clinical Relevance-The smartphone-enabled detection platform realizes the quantitation of biomarker within 10 min, which reveals a valuable potential tool for the early diagnosis of various diseases, even in resource-limited regions.