Pursuing convenient operations and precise testing have become an urgent requirement in clinical diagnosis, treatment, and prognosis. Label-free detection is desirable for obviating the labeling process while maintaining high sensitivity and efficiency. Here, we used the dual properties of highly selective antibody-antigen recognition and potential signaling of biomolecules to construct a label-free electroosmotic flow-driven microchannel (LF-EMB) biosensor based on an antibody-antigen biorecognition-induced charge quenching theory proposed herein. The LF-EMB consists of a one-step immune-reaction, one-button portable device, and supporting microfluidic chip, providing a high-powered tool for rapid on-site testing. The LF-EMB quantified interleukin-6 and kanamycin levels down to 1 pg/mL and 5 pg/mL, respectively, with the whole analysis completed within 35 min. The outstanding sensitivity and detection speed of the constructed LF-EMB provide a convenient option for the quantitative detection of inflammatory markers and antibiotics.