A bioinspired polydopamine (PDA) coating is a good candidate for the rapid and cheap chemical modification of biosensor surfaces. Herein, we report the effect of PDA thickness on the detection sensitivity of a fluorescence biosensor utilizing surface plasmon-enhanced fluorescence. The thickness of PDA films was tuned by the incubation time of the dopamine solution and varied from 1 to 17 nm. The detection sensitivity was evaluated as the limit of detection (LOD) of a fluorescently labelled target analyte by a model immunoassay. The LOD was determined to be 1.6 pM for the thickest PDA film and was improved to 1.0 pM by reducing the thickness to the range from 1 to 5 nm, corresponding to the incubation time of 10 to 60 min. The experimental results indicate that the PDA coating is suitable for the surface functionalization of biosensors in mass production as it does not require precise control of the incubation time.