To date, a range of nanozymes has been reported for their enzyme-mimicking catalytic activity such as solution-based sensors. However, in remote areas, the need for portable, cost-effective, and one-pot prepared sensors is obvious. In this study, we report the development of a highly stable and sensitive gold tablet-based sensor for cysteamine quantification in human serum samples. The sensor is produced in two steps: synthesis of a pullulan-stabilized gold nanoparticle solution (pAuNP-Solution) using a pullulan polymer as a reducing, stabilizing, and encapsulating agent and then, casting the pAuNP-Solution into a pullulan gold nanoparticle tablet (pAuNP-Tablet) by a pipetting method. The tablet was characterized by UV-vis, DLS, FTIR, TEM, and AFM analyses. The pAuNP-tablet exhibited a high peroxidase-mimetic activityviaa TMB–H2O2system. The presence of cysteamine in the system introduced two types of inhibition which were dependent on the cysteamine concentration. By determining Michaelis–Menten's kinetic parameters, we gained mechanistic insights into the catalytic inhibition process. Based on the catalytic inhibition capability of cysteamine, the limit of detection (LoD) was calculated to be 69.04 and 82.9 μM in buffer and human serum samples, respectively. Finally, real human serum samples were tested, demonstrating the applicability of the pAuNP-Tablet for real-world applications. The % R values in human serum samples were in the range of 91–105% with % RSD less than 2% for all replicas. The stability tests over 16 months revealed the ultra-stable properties of the pAuNP-Tablet. Overall, with a simple fabrication method and a novel employed technique, this study contributes to the advancement of tablet-based sensors and helps in cysteamine detection in clinical settings.