The invention relates to an indirect ELISA detection method and kit for a duck adenovirus type 3 antibody and application thereof. The method comprises that steps of coating an enzyme-labeled plate with Fiber2 protein as a coating antigen, incubating a serum sample to be tested with an enzyme-labeled plate after dilution, adding an enzyme-labeled second antibody, incubating and coloring, and measuring the OD value of the reacted serum sample; As that indirect ELISA detection method established by using Fiber2 protein as coat antigen, the duck type 3 adenovirus antibody can be directly detected, and specificity and repeatability are good. The results showed that this method could be used as a method for detection of duck adenovirus type 3 antibody.