Background Pertussis is a highly contagious respiratory disease caused by the bacterium Bordetella pertussis ( B. pertussis ). The infection is difficult to diagnose especially in underserved or resource-limited areas. We developed a low-cost and instrument-free diagnostic method for rapid and accurate detection of B. pertussis on a point-of-care (POC) testing device. Methods We developed a paper/polymer hybrid microfluidic biochip integrated with loop-mediated isothermal amplification (LAMP) method for the rapid and accurate detection of B. pertussis . This microfluidic approach was validated by testing 100 de-identified remnant clinical nasopharyngeal swabs and aspirates, which were confirmed to be either positive or negative for B. pertussis by a validated real-time PCR assay at the Children's Hospital Los Angeles. Findings The instrument-free detection results could be successfully read by the naked eye within 45 min with a limit of detection (LOD) of 5 DNA copies per well. Our optimized bacterial lysis protocol allowed the direct testing of clinical samples without any complicated sample processing/preparation (i.e. DNA extraction) or the use of any equipment (e.g. centrifuges). The validation of the microfluidic approach was accomplished by testing 100 clinical samples. High sensitivity (100%) and specificity (96%) with respect to real-time PCR were achieved. Interpretation This microfluidic biochip shows great potential for point-of-care disease diagnosis in various venues including schools and physician's offices, especially in low-resource settings in developing nations. Funding NIH/NIAID under award number R21AI107415, NIH RCMI Pilot Grant, the Philadelphia Foundation, the Medical Center of the Americas Foundation.