Clostridial diseases significantly threaten livestock health, particularly in cattle, underscoring the need for effective vaccination strategies. This study develops and optimizes a microfluidic-based enzyme-linked immunosorbent assay (ELISA) for the rapid (< 1 h) and cost-effective measurement of IgG antibody levels against various clostridial toxins in cattle vaccinated with a multivalent clostridial vaccine. This assay requires only 5 μL of sample and reagent volume, demonstrating high repeatability and reproducibility with coefficient of variation (CV) values ranging from 0.1 % to 8.5 % across all tested clostridial antigens. The Limit of Detection (LOD) of the assay ranged from 1:150 to 1:800, allowing for sensitive detection of antibody levels. For Clostridium perfringens ε toxin, antibody titers were measured using a commercial kit, while microfluidic ELISA was applied to assess titers against tetanus toxoid, Clostridium septicum α toxin, Clostridium novyi type B toxins, and Clostridium sordellii toxins. Significant increases in IgG antibody levels were observed for C. perfringens ε toxin and tetanus toxoid following both primary and booster vaccine doses, peaking by day 42. Antibody titers against C. septicum α toxins and C. novyi type B toxins increased after the primary dose, peaking at day 42, while no booster effect was seen for C. sordellii. These findings highlight the utility of microfluidic ELISAs as a practical and efficient tool for assessing humoral immune responses to clostridial toxins in vaccinated cattle, with potential application for the herd level disease surveillance and vaccine efficacy assessment.