This study aimed at developing and assessing the fusion proteins with enhanced sensitivity to detect antibodies in plasma as a diagnostic method for tuberculosis. DNA fragments encoding TB16.3 and echA1 gene regions corresponding to proteins TB16.3 and echA1 from Mycobacterium tuberculosis were amplified through PCR. Through a series of restrictions and ligations two novel fusion constructs TB16.3-echA1 and TB16.3-tnPstS1 were produced and expressed in Escherichia coli. These were screened for detection of antibodies in human plasma. The individual antigens TB16.3, echA1 and tnPstS1 and the fusion protein TB16.3-tnPstS1 and TB16.3-echA1 showed sensitivities of 29%, 25.5%, 42.8%, 40.0% and 47.2%, respectively. Lower sensitivity in case of TB16.3-tnPstS1 seems to be due to the structural arrangement between the two proteins, which is likely to mask several of their epitopes. The higher sensitivity of TB16.3-echA1 appears to be due to lesser interaction between the two proteins thus allowing free availability of epitopes for binding antibodies. 64% of TB patients were found positive for either one of the two fusion proteins TB16.3-echA1 and TB16.3-tnPstS1. This study indicates that the novel fusion protein TB16.3-echA1 has a potential in serodiagnosis of TB with improved sensitivity and reliability.