Poultry farming is the most dynamic & 2nd major industry in Pakistan. It has challenges of outbreaks of viral diseases resulting in high mortality, morbidity and huge economic loss in Pakistan. Conventional diagnostic tests are expensive, laborious & require technical expertise. Hence, a highly sensitive & specific dot-ELISA kit is developed, in two steps; first whole antigen is prepared from AIV, NDV and IBV and is applied onto nitrocellulose membrane; second rabbit anti-chicken antibodies is raised, purified and conjugated with enzyme HRP. Dot ELISA results are much comparative with ELISA & have advantages of high specificity, sensitivity & facile. This kit has the advantage of loading multiple antigens from local isolates & can be performed even at poultry forms; with no technical expertise or advance laboratory facilities for interpretations. Even the staff can test their bird’s immune response after vaccination or test for the presence of infection. It can be prepared & used on large scale; this combine approach of several antigens on single strip play a vital role in the diagnosis of poultry diseases or determining immune status and help in controlling diseases in Pakistan.