Visceral leishmaniasis (VL) is a major health problem in the tropical and subtropical regions of the world. The conventional methods for diagnosis of Old World Visceral leishmaniasis are difficult, insensitive, and hazardous. There is no recombinant antigen from old world Leishmania species which can be commercially used for rapid diagnosis. There is an urgent need for a less invasive and accurate method. Here, we report a recombinant antigen from Indian Leishmania donovani for its diagnosis. The kinesin gene of a L. donovani clinical isolate (KE16) from India was PCR amplified for cloning and the immunodominant domain was expressed in Escherichia coli. This recombinant protein or Ld-rKE16 was evaluated for serodiagnosis of Indian kala-azar by ELISA. The recombinant antigen was found to be 100% sensitive and specific for Old World VL cases from India, Pakistan, China, and Turkey. The antigen showed no cross-reactivity with sera from other endemic diseases or healthy controls. The expressed Ld-rKE16 antigen is highly specific and sensitive for diagnosing visceral and post-kala-azar dermal leishmaniasis and is ready for commercialization.