Usually, different assays and instrumentation are required for different types of targets, e.g., nucleic acids, proteins, small molecules, etc., because of significant differences in their structures and sizes. To increase efficiency and reduce costs, a desirable solution is to develop a versatile platform suitable for diverse objectives. Here, we established a versatile detection technique: first, target separation and enrichment were carried out using magnetic beads (MBs); then, different targets were converted to same barcoded DNA strands (BDs) released from gold nanoparticles; finally, sensitive detection of three different targets (miRNA-21, digoxigenin antibody, and aflatoxin B1) was achieved through exonuclease III (Exo III) cyclic cleavage-assisted signal amplification. To simplify the operation, we integrated this technique into a microfluidic chip with multiple chambers in which the requisite reagents were prestored. Just by moving the MBs through different chambers with a magnet, multiple steps can be completed. Due to the limited space in microfluidic chips, the full mixing of MBs and solution is a key point to improve reaction efficiency. The mixing can be achieved by acoustic vibration generated by a small, portable sonic toothbrush. Based on the microfluidic chip, the detection limits of the above three targets were 0.76 pM, 0.16 ng/mL, and 0.56 nM, respectively. Furthermore, miRNA-21 and Digoxigenin antibody (Dig-Ab) in serum and AFB1 in corn powder were also used to demonstrate the performance of this chip. Our versatile platform is easy to operate and is expected to develop into an automatic "sample-to-answer" device.