In this study, an immunosensor based on a graphene oxide (GO)-functionalized microfiber is developed and used for the analysis of rabies virus (RV) immunodetection. For this, first, a standard single-mode fiber was discharged through an optical fiber fusion splicer to form a double-tapered fiber, after which the double-tapered fiber was fused and tapered to produce a high-sensitivity microfiber. Subsequently, the microfiber surface was modified with GO, and RV antigens were immobilized on the surface of the sensor for the immunoassay of RV antibodies. The experimental results indicate that the detection range of the biosensor for RV antibodies is 200 fg/mL^-1 ng/mL, its detection of limit (LOD) is approximately 225.56 fg/mL, and its detection sensitivity and dissociation coefficient are approximately 1.099 nm/log (mg·mL^-1) and 2.92×10^-11 M, respectively. When the sensor is used in control experiments and clinical immunoassays for other antibody solution samples and RV-positive serum, it yields weak responses to the former and significant responses to the latter, indicating its high specificity to RV antibodies. The proposed immunosensor based on the GO-modified microfiber demonstrates the advantages of facile fabrication, micro-nano size, high sensitivity, and low cost.