Dengue has become endemic in Pakistan, and increasing cases of dengue have been reported every year since 2006. The objective of the study was to serologically and molecularly diagnose dengue disease and to identify the circulating serotypes in patients of the Lahore region. Total 345 blood samples were collected from Jinnah Hospitals Lahore (JHL) and Mayo Hospital Lahore (MHL). Dengue specific antigen and antibodies were detected in samples by two serological methods namely, immunochromatographic test device (ICT) and Enzyme-linked immunosorbent assay (ELISA). Acute phase samples were further subjected to molecular detection and serotyping of dengue virus by nested reverse transcriptase-polymerase chain reaction (RT-PCR). Similarity and divergence studies and the phylogenetic relationship was conducted on obtained sequences. Out of 345 samples subjected to serological analysis, 114 (33%) were positive to dengue by ICT method while 110 (31.8%) were detected positive by ELISA. Total 108 (31.3%) samples were confirmed dengue positive by both serological methods, out of which 39 (36.1%) samples were NS1 positive by both ICT and ELISA methods. By nested RT-PCR of 39 NS1 positive samples, the dengue virus was detected in 8 (20.5%) patients. In this study DENV-1 was detected in 1(12.5%) sample, DENV-2 in 3 (37.5%) samples and DENV-3 in 4 (50%) samples out of 8 PCR positive samples. The DENV-3 was the prevalent serotype. Similarity studies and phylogenetic analysis conducted on three serotypes (DENV-1, DENV-2 & DENV-3) revealed their relatedness with dengue isolates from Sri Lanka, Singapore and India. This study demonstrated the prevalence of DENV-3 serotype in Lahore region in 2013 that is unique from the previously reported dengue serotypes in Pakistan. Moreover, this study concluded that both selected serological methods (ICT and ELISA) are equally good and comparable. The diagnostic frequency could efficiently be increased by detecting NS1 antigen along with IgM/IgG antibodies.