Fluorene is a polycyclic aromatic hydrocarbon, which is a hazardous toxic chemical in the environment. The measurement of low concentrations of fluorene is a subject of intense interest in chemistry and in the environment. Polypyrrole chitosan cobalt ferrite nanoparticles are prepared using the electrochemical method. The prepared layers are characterized using field emission scanning electron microscopy, Fourier transform infrared spectroscopy, and energy dispersive spectroscopy. The layers are used to detect fluorene using the surface plasmon resonance technique at room temperature. The composite layer is evaluated after detection of fluorene using atomic force microscopy. The fluorene is bound on the layer, and the shift of the resonance angle is about 0.0052∘, corresponding to the limitation of 0.01 ppm.