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How to balance the sensitivity and signal-to-noise ratio of immunosensor remains many challenges during various diseases diagnosis. Here we develop a new microfluidic immunosensor based on surface-modified mesoporous nanofibers, and simultaneously realize an ultra-sensitivity and high signal-to-noise ratio for the detection of multiple biomarkers. In the current study, we fabricated titanium dioxide (TiO 2)-based mesoporous electrospinning nanofibers, and modified nanofiber surface with both octadecylphosphonic acid (OPA) and poly(ethylene oxide)-poly(propylene oxide) triblock copolymer (PEO-PPO-PEO). Such nanofibers as solid substrate are covered on microfluidic channels. The porosity of our nanofibers dramatically increased the adsorption capability of antibodies, realizing an ultra sensitivity of biomarker detection. PEO-PPO-PEO modification can significantly block non-specific absorptions, obtaining a satisfied signal-to-noise ratio. For the detection of HIV p24 and interleukin 5 (IL-5), our immunosensor increased 6.41 and 6.93 fold in sensitivity and improved 504.66% and 512.80% in signal-to-noise ratio, in compared with gold standard immunoassay (ELISA) used in the clinic. Our immunosensor also broaden the linear range for the detection of HIV p24 (0.86–800 pg/ml) and IL-5 (0.70–800 pg/ml), in compared with ELISA which is 5.54–500 pg/ml for HIV p24 and 4.84–500 pg/ml for IL-5. Our work provided a guideline for the construction of advanced point-of-care immunosensor with an ultra-sensitivity and high signal-to-noise ratio for disease diagnosis. • A new immunosensor can realize a high-quality biomarker binding and avoid the non-specific absorption from interfering substances. • A new immunosensor can satisfy ultra-sensitivity and high signal-to-noise ratio for the detection of multiple biomarkers. • A new immunosensor can combine with microfluidic chips for realizing a high-quality point-of-care testing. [ABSTRACT FROM AUTHOR]