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We report a self-sufficient microfluidic paper-based lateral flow immunoassay device (μLFD) for highly sensitive detection of the thyroid-stimulating hormone (TSH). Fabrication of the paper microchannels involves engraving the nitrocellulose membrane with a CO2 laser to create narrow flow paths, which constrain the fluid flow over the test zone. The proposed microchannel modified devices were studied for detection of the TSH using gold nanoparticles as labels. The effect of such microchannel modified LFDs has led to an improvement in sensitivity by nine times and the limit of detection by 6.6 times due to the slow flow rate of the sample compared with the traditional LFD. In addition, the binding of gold nanoparticles over the test line is more uniform in the case of the μLFD, thus minimizing leading-edge effects, resulting in more accurate quantitative analysis. The proposed strategy offers great potential for multiplex detection of biomarkers with increased sensitivity without introducing any hydrophobic materials to the LFD.