Introduction: The detection of Mycobacterium avium subsp. paratuberculosis (MAP) in biological samples has been hampered because of the lack of reliable antibodies able to specifically recognize components of its cell wall. The subspecies MAP produces specific lipopentapeptide L5P or lipotripeptide L3P on the cell wall surface, which has not been yet found so far in other mycobacterial species. Purpose: Development of single-chain antibodies (scFvs) for a rapid identification of MAP targeting the L5P antigen. Methodology: Phage display technology used a phage library of scFv antibodies to screen antibodies against the cell wall L5P extracted from MAP strain K-10. The scFv termed L7 was produced as a recombinant protein in E. coli and purified by affinity chromatography. The portion of the L5P molecule that binds the antibody was determined by exposing the antibody to seven derivatives of the L5P using fluorescent microscopy. Furthermore, the cross-reactivity of the antibody with other mycobacterial species, such as M. abscessus, M. avium subsp. avium, M. bovis, and M. smegmatis were assessed by immunostaining. Results: L7 was successfully produced as a soluble recombinant protein in E. coli. No cross-reaction was observed when L7 was exposed to other mycobacterial species, suggesting that L7 is specific to MAP. Furthermore, binding analysis of various L5P analogs showed that L7 recognizes only the pentapeptide portion of the molecule containing the amino acid sequence D-Phe-L-NMe-Val-L-Ile-L-Phe-L-Ala, and not the lipidic moiety or the tripeptide sequence of the L3P present on the sheep type MAP cell wall. Lastly, the antibody was able to identify MAP in milk, colostrum, and biopsies by immunofluorescence.