Introduction : Perfect and well-organized diagnosis of dengue is significant for medical concern, observation maintenance, studies about pathogenesis, and research for vaccines. Diagnosis is also significant for infection verification (DF or DHF/DSS), to distinguish dengue from other infections, and further flavivirus diseases, and for the designated treatment strategy organization as well as estimation of persons having severe infection (1). Materials & Methods : There were 85 serum samples and 03 whole blood samples collected from suspected dengue patients, from different hospitals and laboratories of Karachi city in year 2012. Initially three whole blood samples were subjected to Immuno fluorescent staining technique then RNA was extracted from dengue serum samples and subjected to one step multiplex Real Time RT PCR and 2% agarose gel respectively. Results : All the 85 serum samples gave positive results on one step multiplex Real Time RT PCR and also gave bands on 2% agarose for dengue serotype 2, 3 and 4. Immuno fluoresent staining was also positive for dengue serotype (2). Conclusion : The results of this study shows that both immunofluorescence technique as well as one step multiplex Real Time RT PCR are efficient and rapid techniques for diagnosis and for research purpose equally, if it would be fully establish in local setting. Along with these finding we also found that the major dengue virus infections in year 2012 were of dengue serotype 4, while dengue serotype 2 and dengue serotype 3 were present in very small percentage i.e., 10% and 6% respectively.