The present study was envisaged with an aim to evaluate the sero-positivity of Trypanosoma evansi antibodies in Indian elephants by using card agglutination test for trypanosomes (CATT/ T. evansi) and to assess haemato-biochemical alterations in positive cases. The blood samples were collected from ear veins of elephants (n = 12), kept at Mahendra Chaudhury Zoological Park, Chhatbir, Punjab. Agglutination patterns were scored as − (negative) and +, ++ or +++ (positive). One sample from female elephant was found strongly positive with +++ agglutination pattern, whereas one sample of male elephant was observed slightly positive (+). Significant hyperproteinaemia (p < 0.01), hyperglobulinemia (p < 0.05), leucocytosis (p < 0.05), and hyponatremia (p < 0.01) were amongst the haemato-biochemical alterations observed in sero-positive individuals. Molecular confirmation of T. evansi was also performed using polymerase chain reaction targeting VSG (RoTat 1.2) gene. The present study highlights the first record of card agglutination-based assessment of T. evansi in captive wild animals in India and also promulgates the haemato-biochemical responses in T. evansi sero-positive elephants. [ABSTRACT FROM AUTHOR]