Reliable and cost-effective methods for thiamine quantification are essential in clinical diagnostics and nutritional monitoring. Traditional techniques, such as HPLC and ELISA, have limitations in terms of cost, sensitivity, and technical complexity. This study introduces the Enzyme-Labeled Protein-Ligand Assay (ELPLA), a novel method utilizing thiamine-binding protein (TBP) for the sensitive and specific measurement of thiamine levels in serum. TBP was purified from mung beans and quantified using a bovine serum albumin (BSA) standard curve, achieving a coefficient of determination (R² = 0.9999). We tested its stability under varying temperatures and chemical conditions. ELPLA was optimized using specific concentrations of TBP and a thiamine-avidin conjugate, with evaluation of its precision, accuracy, and linearity. The method was compared with HPLC and ELISA in serum samples from three groups: healthy individuals, patients with type 2 diabetes mellitus (T2DM), and chronic alcohol users. Our findings showed that TBP maintained good thermal stability at-20 °C with optimal binding at a pH of 7.5. ELPLA exhibited remarkable precision (coefficient of variation, CV = 2.85%) and recovery rates (88–104%), with linearity remaining consistent even after serial dilution. Results correlated well with HPLC (r = 0.992) and ELISA (r = 0.861). Both ELPLA and HPLC detected significant differences in serum thiamine levels among the groups, while ELISA displayed limited sensitivity. Overall, ELPLA stands out as a robust, sensitive, and cost-effective alternative to existing methods. Its simplicity and reliability make it suitable for routine clinical and nutritional use, particularly in low-resource settings. Further validation across various biological matrices and food systems is recommended. © 2025, Editorial board of Journal of Experimental Biology and Agricultural Sciences. All rights reserved.