Infectious bronchitis virus (IBV), an avian coronavirus, significantly affects the performance of both the egg-laying and meat-type birds causing the foremost of economic loss in poultry industry. This paper aims to develop a rapid, low-cost, and sensitive biosensor for IBV detection by using molybdenum disulfide (MoS 2). MoS 2 is a 2-D nanosheet which has strong high fluorescence-quenching ability when applied to a dye-labeled antibody (Ab). In this paper, we developed an Ab-functionalized MoS 2-based fluorescent immunosensor, which utilized the fluorescence resonance energy transfer (FRET) between the MoS 2 and fluorescence dye during the Ab–antigen interaction. The assay was performed on a low-cost cotton thread-based microfluidic platform due to the good wicking property and flexibility. Upon the optimization of assay conditions, the immunosensor demonstrated remarkable sensitivity of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$4.6\times 10^{2}$ \end{document} EID 50 per mL and specificity with a dynamic linear response range of 10 2–10 6 EID 50 per mL for IBV standard solutions. The developed immunoassay successfully detected the IBV spiked chicken serum with satisfactory results. The foregoing presents its potential application for on-farm detection.