Enterotoxigenic Escherichia coli (ETEC) causes diarrhea in both humans and animals. The contaminated food and water are the most common vehicles for ETEC infection. The colonization factor antigen (CFA-1) is a fimbriae protein that promotes adherence of the ETEC strain to the epithelium of the small intestine of the host. In this study IgY proteins were produced against the CFA-1 of ETEC in immunized white leg horn chickens. The collection of antibody was found higher in 25% and 50% ammonium precipitated samples. The isolated CFA-1 protein (0.5 mg/ml) was purified by dialysis (0.42 mg/ml), gel-filtration (0.08 mg/ml), and ion-exchange chromatography (0.065 mg/ml). The CFA-1 protein obtained was submitted to pBLAST which matched with CFA-b with accession no.-AAC41415.1. The CFA-1 gene was cloned in pUC18 vector, and transformed into Escherichia coli DH5α strain. In conclusion, we have successfully purified and characterized monoclonal antibodies (IgY proteins) against CFA-1 of enterotoxigenic E. coli that may be useful for diagnosis of widely occurring Escherichia coli infections.