The objective of the studies was development and use of a customized ELISA kit to diagnose mycoplasmal infections in poultry. The frozen strain of S-6 M. gallisepticum, concentrated and ultra-sounds processed, served as the antigen. The best extinction (OD) was noted with the antigen concentration of 100 µg/ml, sera diluted at 1:100 and conjugate diluted at 1:250. It was best to preserve antigen coated plates overnight at 4°C. The ELISA was used to detect specific anti-M. gallisepticum antibodies both in chickens experimentally infected and in hens and turkeys naturally infected with this microorganism. Basing on the calculated value S/P it was possible to compare the sensitivity of the ELISA with an agglutination test (SPA) and a hemagglutination inhibition test (HI). The value of S/P for positive sera was more than 0.5, while for negative sera, below 0.5. The value of S/P in the ELISA for sera of chickens experimentally infected with high positive titres in HI (320 and 640) was more than 2.5, and for sera of HI titre 80 these values of S/P ranged from 1.0 to 1.5. The investigation of naturally infected birds showed that S/P in the ELISA test for sera of full agglutinating properties (3+) was more than 2.0, and for individual sera it was up to 3.5. However, weakly positive sera in the SPAS have a negative or slightly positive S/P value (from 0.5 to 1.0). The ELISA test used to detect antibodies specific to M. gallisepticum proved to be more sensitive than the SPA and HI test.