In this work, we describe an optical fiber immunosensor for on-site bioaerosol monitoring application. Considering its integration to the monitoring system, a polymethyl methacrylate (PMMA) fiber was used as a sensor probe. The fiber edge surface was modified with a layer-by-layer (LbL) assembled film for immobilizing primary antibodies. The LbL film was formed by depositing alternating layers of cationic and anionic polymers, namely, poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA), respectively. To assess the sensor performance, a model immunoassay experiment was conducted with mouse (mIgG) and horseradish peroxidase (HRP)-labeled anti-mouse (a-mIgG) antibodies. mIgG was immobilized on PAAs in the LbL film. After affinity binding of HRP-labeled a-mIgG to mIgG, a solution containing a fluorescent substrate, 10-acetyl-3,7-dihydroxy-phenoxazine (ADHP), is added. The resultant fluorescent product, resorufin, is then excited with light of 570 nm wavelength from the fiber edge, and the emitted fluorescence of 600 nm wavelength is collected by an identical fiber edge and detected by a photomultiplier tube. As results of the experiment, a sensor limit of detection (LOD) of 85 ng･mL−1 and a dynamic range of 0.71–100 µg･mL−1 for a-mIgG were determined. It was demonstrated that the optical fiber immunosensor has superiority in size and flexibility with high sensor performance, and thus can be easily incorporated into a monitoring system.