In clinical diagnosis, accurate and reliable measurement technologies for the detection of disease biomarkers at ultralow concentrations can provide guidance for the initiation of treatment and potentially improve survival for patients. Here, we demonstrate an optical microfiber reader for enhanced analytical sensitivity in enzyme-linked immunosorbent assays (ELISA) that enables the detection of tiny changes of the refractive index (RI) induced by the catalyzed oxidation of substrate, owing to the strong interaction between the evanescent field and surrounding medium. By employing the microfiber reader for the C-reaction protein (CRP) and interleukin-6 (IL-6) assays after the enzymatic signal amplification in ELISA, we experimentally investigate the biosensing capacity of the device. As a result, log-linear relations of CRP and IL-6 detection in PBS and human serum between the concentration and spectral response were obtained at both nanogram and picogram levels, respectively, and anti-CRP/HRP detection as low as 9.75 pg/mL was achieved, which was undetectable by the conventional spectrophotometry. With a stable, accurate, and color-free detection capacity, this optical microfiber reader has a promising prospect in early disease diagnosis and clinical treatment.