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There are specific laboratory tests available for the diagnosis of Covid-19 and dengue, during the present pandemic era of prioritized focus on Covid-19 assessment; there are possibilities that persons with dengue may remain undiagnosed. The Purpose of the present study explores the role of biochemical markers in the differential diagnosis of Covid-19 and dengue. For this study a total of 212 participants with Acute Febrile Infection were Diagnosed forCovid-19 and dengue at Hayatabad Medical Complex Peshawar Kp Pakistan. The Covid-19 and dengue identification were performed using standard tests followed by hematological profiling which included neutrophil lymphocyte ratio (NLR), platelet count, Vitamin D3 Assessment, SGOT, SGPT and SPO2 concentration levels. The Result of our research show that out of 212 Individual, 118 were diagnosed with Covid-19 positive only, 18 dengue positive only, 5 co- infected with Covid-19 and dengue, and 71 persons with Acute Febrile Illness (control group). ANOVA Test result show that means SPO2 was significantly lower in Covid-19 and dengue than control, while SGPT and SGOT levels of Covid-19 and dengue patients were significantly higher than the control group. . The mean NLR was significantly higher in Covid-19 and dengue than control and Vitamin D3 levels were significantly reduced for Covid-19 patients. Besides, thrombocytopenia was observed only in dengue patients. The results advocate the potential use of combinations of these makers in differential diagnosis of these two fatal viral conditions and can help by enabling the adaptation of the therapeutic conduct to the needs of individual patients.