ABSTRACT: Enzyme-linked immunosorbent assay (ELISA) is a popular technique for quantifiable detection of specific antibodies in warm-blooded animals, but it has not been accepted for detection of fish antibodies because of its low reproducibility, which is due in part to high background optical density (OD) measurements. In the present study, we report that the high background of a fish antibody-detection ELISA resulted from non-specific adsorption of fish immunoglobulin M (IgM) to blocking reagents on the ELISA plate wells. Four fish sera (from rainbow trout Oncorhynchus mykiss, masu salmon O. masou, Japanese flounder Paralichthys olivaceus and koi Cyprinus carpio) were poured into ELISA plate wells pre-blocked with several blocking reagents (skim milk, soybean milk, bovine serum albumin, fetal bovine serum, gelatin and Roche BlockingReagent^®) and then washed out in order to measure the remaining fish IgM on the ELISA plate wells. Significant amounts of fish IgMs (OD absorbance at 492 nm: 0.3 to 1.1) remained on the ELISA plate wells with no antigenic protein except blocking reagents. The amount of remaining fish IgMs on the ELISA plate wells decreased significantly following treatment of fish sera with skim milk. However, the specific immuno-reactivity of fish IgM was not reduced by such treatment. Thus, we conclude that treatment of fish sera with skim milk is useful in reducing the high background OD often observed in fish IgM detection ELISA.