Gold nanoparticles (AuNPs) have attractedthe interest of scientists owing to their unique applications in diverse fields. This study presents a report on the utilisation of glutaraldehyde (GA) as a reducing and stabilising agent for the one-pot preparation of AuNPs functionalised with GA (GA-AuNPs), designed specifically for the immobilisation of proteins. The preparation of stable GA-AuNPs has been systematically explored by varying the temperature, quantity of GAs, and pH. Subsequently, bovine serum albumin and horseradish peroxidase have been immobilised on the GA-AuNPs surface to demonstrate the versatility of GA-AuNPs. Dynamic light scattering results indicated that the prepared quasi-spherical GA-AuNPs exhibited a zeta potential of −31.2 ± 2.3 mV and had a size of 5.7 ± 0.5 nm. The advantages of this method include its simplicity at room temperature under approximately neutral pH conditions, elimination of the need for additional steps, and streamlining of the immobilisation process. These results emphasise that GA-AuNPs are promising candidates for effectively transporting proteins or enzymes across a diverse range of applications.