Dengue fever is a viral disease transmitted by mosquitoes that continues to pose a serious public health threat worldwide, infecting millions annually. Nearly 40% of the global population remains at risk. Thus, early and precise diagnosis is crucial for monitoring the spread of the disease and ensuring appropriate medical care. Here, we report serotyping of infecting DENVs and natural anti-dengue IgG antibodies in total of 207 DENV-infected serum specimens, of which 95 and 112 dengue suspects’ sera collected from Dhaka Medical College Hospital and Chittagong Medical College Hospital, respectively. 35 out of 95 samples from Dhaka were NS1 positive, whereas 17 samples were tested positive by RT-PCR. It was noted that all 17 samples were single DENV serotype, where DENV4 constituted the highest prevalence (35%), followed by DENV2 (29%), DENV1 (24%) and DENV3 (12%). On the other hand, 42 out of 112 NS1 positive samples collected from Chittagong Medical College Hospital were DENV positive by RT-PCR. Among them, the single DENV serotype was 76%, and multiple DENV serotypes were 24% of which DENV4 was also the most prevalent DENV serotype. Investigation of natural anti-dengue IgG antibodies in DENV-infected sera indicated that natural DENV infections resulted both serotype-specific and sero-cross-reactive anti-dengue antibodies. The correlation of infecting DENV serotype and natural anti-dengue IgG and IgM antibody response between Dhaka and Chittagong has been reported in this study. Our analysis revealed that dengue-infected samples from Chittagong presented a more critical situation compared to those from Dhaka, as 24% of the samples demonstrated concurrent infection with two or more DENV serotypes, whereas the Dhaka cases were associated with infection by a single serotype.