In acidic environments, acid phosphatases (EC 3.1.3.2) play a crucial role in hydrolyzing phosphate ester linkages. Two isoforms of Acid phosphatases namely AP-I and AP-II were purified to homogeneity from the seeds of Erythrina indica using a combination of gel filtration and affinity chromatography techniques. The purification process involved multiple steps to ensure the enzymes were free from other seed components, thereby facilitating detailed characterization. We report in this study the active site characterization of acid phosphatase form AP-I. AP-I active site of the purified AP-I was characterized in detail through chemical modification studies, which revealed the presence of one residue each of carboxylate, tryptophan, and serine amino acid. Substrate protection experiments using p-nitrophenyl phosphate effectively prevented the modification of all three residues, suggesting their essential role in the enzyme's active site. These experiments provided strong evidence that these residues are directly involved in the catalytic process. Kinetic studies of the partially inactivated enzyme, achieved through the use of specific modifying agents Dicyclohexylcarbodiimide (DCCD) for carboxylate, N-Bromosuccinimide (NBS) for tryptophan, and Phenylmethylsulfonyl fluoride (PMSF) for serine, further confirmed the involvement of these residues in the catalytic mechanism. The results demonstrated that the inactivation of any of these residues significantly impaired the enzyme's activity, highlighting their critical roles in the catalytic process. The results provide a comprehensive understanding of the active site architecture and the catalytic mechanism of AP-I function.