Herein, we report a strategy for quantifying the relative proportion of di-block copolymers of poly(ethylene glycol) (PEG) on fluorescein isothiocyanate (FITC) functionalized gold nanoparticles (AuNP) by releasing the FITC via the self-assembly process. The self-assembly processes of terminally functionalized trithiocarbonate PEG and homoglycopolymers were also assessed to demonstrate the usefulness of the terminal functional groups of the glycopolymers on the surface of well-dispersed colloidal suspensions of AuNP. The synthesized AuNP colloidal suspension showed a surface plasmon resonance (SPR) peak at 517 nm and a red shift of about 19 nm, observed after the addition of FITC to the AuNP suspension. The transmission electron microscopy (TEM) images and histogram of the AuNP nanoparticles size distribution showed an average diameter of 8 nm, where the FITC functionalization of AuNP led to agglomeration. The fluorescence spectra reveal that the PEG-I binds to the FITC–AuNP and subsequently, FITC is proportionately released from the FITC–AuNP surface. This indicates that PEG-I possesses a stronger binding affinity for AuNP. Released concentrations of FITC fluorescence were calculated based on the amount of PEG-I added to the colloid of FITC–AuNPs and a linear response was observed. The homoglycopolymer, poly[6′-(acryloxy)hexyl-2,3,4,6-tetra-O-acetyl-D-glucopyranoside] [P(AHTAGP)], showed a weak binding affinity towards the AuNP surface, which was significantly improved in the PEG based glycopolymer, PEG-b-P(AHTAGP), with similar functional moiety. The di-block copolymer with glucose moiety of greater pendant spacer length showed better binding affinity for AuNP, whereas the bi-ligand functionalization of AuNP with the glycopolymer and FITC was more effective with glucose moieties of shorter pendant spacer length. The variation of the pendant spacer length of the functional moiety in the di-block glycopolymers has the potential advantage of serving multiple purposes in targeted delivery and detection.