Lateral flow biosensor (LFB) is one of the most successful and applied commercial detection methods for food safety, drug abuse, and disease. Here, we integrated the ultrasound enrichment as sample preparation with LFB to achieve the ultra-trace protein detection in blood. When the ultrasound field is applied, the interaction between the acoustic field and gold nanoparticles can gather specifically modified gold nanoparticles toward pressure nodes in seconds and enrich target proteins. Such an approach can detect protein with a linear range of 1-20 ng mL-1 and detection limit of 0.58 ng mL-1 in blood within 20 min, which enormously reduces false positive readings caused by interference in real blood samples with complex components. Such a microchip that integrated acoustic enrichment with LFB shows great potential in detecting ultra-trace biomarkers for clinical diagnosis.