Background : Glycogen is the cyanobacterial reserve carbohydrate which is currently the focus of many studies. However, quantification of intercellular glycogen needs thorough investigation. The hypothesis is that glycogen can bind to nanogold. This binding can be used as an important tool for the quantification of intracellular glycogen. Methods: Two strains of cyanobacteria were demonstrated to biosynthesise nanogold intracellularly and to bind to cellular glycogen. Then, spherical gold nanoparticles were chemically prepared and tested for binding to the glycogen molecule of cyanobacterial strains; Lyngbya majuscula and Cyanothece sp. via biochemical method. Experimental analyses were conducted to determine the morphological and optical properties of the Au–glycogen hydrocolloids, together with the analysis of the absorption spectra. The luminescence emission of AuNPs that resulted from recombination between electron in excited state HOMO and hole in ground state LUMO of gold nanoparticles according to Mie theory was recorded. The size diameter and shape of AuNPs were measured via scanning electron microscope and dynamic light scattering techniques. The stability of Au-glycogen was studied by the sequential addition of standard solutions of glycogen in the concentration range (10–100 µmol l− 1) into the prepared AuNPs colloidal solution by recording the SPR and luminescence intensity of AuNPs. Results: The color of the cyanobacterial strains turned into purple color that indicated the formation gold nanoparticles inside the cell (intracellularly). To confirm binding between nanogold and glycogen, the absorption spectrum of AuNPs-glycogen showed plasmon band that was centered at 520–540 nm, suggesting that gold nanoparticles were attached to the surface of the glycogen particles. The interaction of the gold nanoparticles with the biopolymer was further confirmed by photoluminescence spectroscopy analysis. The size diameter of the Au-glycogen in both Lyngbya majuscula and Cyanothece sp. were observed to be 41.7 ± 0.2 nm and 80 ± 30 nm, respectively. FTIR analysis showed that the glycogen absorption peak was observed at 1,000 to 1,200 cm− 1 and exhibited an increase corresponding to the increase in glycogen content in both cyanobacteria. In cyclic voltammetry scans, the Au3+/Au0 redox coupling was observed in case of Lyngbya majuscula indicating the formation of AuNPs-glycogen but in Cyanothece sp. the oxidation anodic peak of AuNPs disappeared which indicated that the AuNPs were highly stabilized in Lyngbya majuscula rather than in Cyanothece sp. This may be attributed to the presence of many thiazole peptides in Lyngbya majuscule. The luminescence of AuNPs showed more stability by the addition of gradual concentrations of glycogen and stronger emission of AuNPs as glycogen protected AuNPs agglomeration. The validation method applied to detect the concentration of glycogen was the use of the change in luminescence of AuNPs in correspondence to binding with glycogen. The detection limit (LOD) and quantitation limit (LOQ) were observed to be 0.89 and 2.95 µmol L-1 respectively. Correlation convention (R) was 0.995. The good chemical stability of this colloidal system and the glycogen biomolecules are studied via density functional theory (DFT). The HOMO level of glycogen unit was closed near to LUMO level of Au3+ that supported the bioconversion of Au3+ into AuNPs via glucose units of glycogen. The detection limit (LOD) and quantitation limit (LOQ) were observed to be 0.89 and 2.95 µmol L− 1 respectively, with R (correlation convention) equal to 0.995. Computational calculations such as density functional theory (DFT) was used to confirm the Au-glycogen complex in bio-system. The HOMO level of glycogen unit was closed near to LUMO level of Au3+ that supported the bioconversion of Au3+ into AuNPs via glucose units of glycogen. Conclusion: The associations formed between the gold nanoparticles and glycogen resulted in good chemical stability. The aggregation of the gold nanoparticles is related to the glycogen concentration and has a profound influence on the absorption properties of Au-glycogen systems. The interparticle distance between AuNPs and glycogen molecule induced the shift in the plasmon band.