Bacterial vaginosis is one of the most frequent vaginal infections. Its main etiological agent isGardnerella vaginalis, which produces several virulence factors involved in vaginal infection and colonization, in particular, sialidase (SLD), a potential clinical biomarker that participates in immune response modulation and mucus degradation. The main objective of this work was the production and evaluation of a monoclonal antibody againstG. vaginalissialidase and its validation in immunoassays. For immunization of mice, a synthetic multiantigenic peptide was used, and hybridomas were generated. After fusion, hybridomas were evaluated for antibody production and cloned by limited dilution. One clone producing IgG1 was selected and characterized by indirect ELISA, dot blot, and Western blot, and we also tested clinical isolates and HeLa cells infected withG. vaginalis. The results showed that the anti-SLD antibody recognized a single protein of ~90 kDa that correlated with the estimated molecular weight of SLD. In addition, anti-SLD antibody recognized SLD from complete bacteria and from culture supernatants of infected Hela cells. In conclusion, our results showed that the anti-SLD antibody recognized SLD from different sources and could be considered a new tool for the diagnosis of bacterial vaginosis.