It is of paramount importance to be able to accurately quantify surface coverage of antibodies on gold nanoparticles (AuNP) so as to optimise the sensitivity of AuNP-based immunosensors. Herein, we developed a fluorescence-based method to directly quantify rabbit immunoglobulin G (IgG) used as antibody model bound to AuNP. Rabbit IgG was first labelled with fluorescein-5-isothiocyanate (FITC) prior to conjugation to AuNP via either physisorption or chemisorption. IgG-conjugated AuNP were treated with NaCN to dissolve the AuNP and restore the fluorescence emission that was quenched in the presence of the metallic colloids, followed by quantification of fluorescein by spectrofluorimetry. This direct assay gave about 4 IgG bound to each 15-nm diameter AuNP for both immobilization strategies. This surface coverage value was in good agreement with that determined from the theoretical value calculated from the Localized Surface Plasmon Resonance (LSPR) band shift. For comparison, we also applied two indirect methods based on the quantitation of excess IgG remaining in the supernatant using fluorescence assay or enzyme-linked immunosorbent assay (ELISA). The indirect assays, either fluorescence or ELISA, commonly used to assess the antibody coverage on AuNP, overestimated the IgG surface coverage to a large extent, since up to 3 to 4 times higher coverages were measured. Therefore, the direct fluorescence method reported in this paper appears as a valuable method for quantification of surface coverage of antibody on AuNP.