Citrus is one of the most popular fruit crops of the world. It is cultivated in around 140 countries, including many in the Asia-Pacific. During recent years, there has been a significant increase in the citrus production mainly on account of its increased use as a nutritious and health drink; however, with increasing area, its production has not increased because of several reasons including diseases and pests. Among all diseases, Phytophthora causes 80% to 90% economic losses to grower. It has an extensive host range infecting different crops and weeds. For better control management, there is a need of correct diagnosis. The present study was designed to develop correct diagnosis methods. Soil samples were collected from an infected field from different locations. From the samples, Phytophthora was isolated and purified using PARPH-CMA medium. The rDNA was amplified using the universal primer, i.e., ITS, and the amplicons were sequenced, and further primers were designed using primer-3 and validated using the PCR method; we found a set of primer that can detect only Phytophthora from soil and infected root samples. Antiserum was raised using rabbit and used for standardization of ELISA. The titer was fixed at 1:50 of antigen, 1:16000 of primer antibody, and 1:2000 of secondary antibody dilution. While proceeding with work, we were able to diagnosis lowest density ofPhytophthora in the field. As well as using this approach field soil had been tested for presence of phytophthora. This diagnosis method helps in the identification and screening of diseased planting material from nursery. For better orchard, there is a need for soil as well as planting material testing for disease control management. Thus, this is very quick, accurate and sensitive method for the detection of Phytophthora, which is a national problem for citrus growing industry.