Cystic echinococcosis (CE) is a neglected tropical zoonotic disease mostly prevalent in tropical and subtropical region of the world. The diagnosis of the disease is difficult and expensive and could not differentiate in current and past infection with higher cross reactivity. Hydated cyst fluid is a mixture of various excretory and secretory antigens which can be used for the development of an effective, most specific and sensitive diagnostic assay like Dot-ELISA. Hydated Cyst (HC) from slaughter sheep were collected and Hydated Cyst Fluid (HCF) were rinsed through a sterile syringe. Sodium Dodecyl Polyacrylamide Gel Electrophoresis (SDS-PAGE) were used for the separation of various antigenic peptides followed by identification and characterization of most suitable antigenic peptide for the development of Dot-ELISA assay. a total of five different peptides of EgAgB having different bands sizes including 10-12 kDa, 22-24 kDa, 38-40 kDa, 70-75 kDa and 170-173 kDa were isolated from hydated cyst fluid (HCF) of sheep through SDS-PAGE analysis. The evaluation of entire separated bands for Dot-ELISA development showed that the EgAgB peptide of 22-24 kDa could work as a promising candidate for dot-ELISA development. The developed dot-ELISA procedure had a diagnostic sensitivity of 96.4% and sensitivity of 98.6% for diagnosis of the condition in human hydated cyst patients. In comparison to the commercially available ELISA kit which shows a sensitivity of 85.7% and specificity of 97.2% the current developed dot-ELISA Kit will be a better alternative with higher efficacy in human diagnosis. The current study concluded that HCF contain various antigenic peptides among which the antigenic peptide of 22-24kda band size could be used for the diagnosis of CE infections in under-developed and developing countries