The lateral-flow immunoassay (LFA) is an inexpensive point-of-care (POC) paper-based diagnostic device with the potential to rapidly detect disease biomarkers in resource-poor settings. Although LFA is inexpensive, easy to use, and requires no laboratory equipment, it is limited by its sensitivity, which remains inferior to that of gold standard laboratory-based assays. Our group is the only one to have previously utilized various aqueous two-phase systems (ATPSs) to enhance LFA detection. In those studies, the sample was concentrated by an ATPS in a test tube and could only be applied to LFA after it had been extracted manually. Here, we bypass the extraction step by seamlessly integrating a polyethylene glycol-potassium phosphate ATPS with downstream LFA detection in a simple, inexpensive, power-free, and portable all-in-one diagnostic device. We discovered a new phenomenon in which the target biomarkers simultaneously concentrate as the ATPS solution flows through the paper membranes, and our device features a 3-D paper well that was designed to exploit this phenomenon. Studies with this device, which were performed at room temperature in under 25 min, demonstrated a 10-fold improvement in the detection limit of a model protein, transferrin. Our next-generation LFA technology is rapid, affordable, easy-to-use, and can be applied to existing LFA products, thereby providing a new platform for revolutionizing the current state of disease diagnosis in resource-poor settings.