The World Health Organization (WHO) has set forth a global call for eradicating malaria, caused majorly by the protozoan parasites Plasmodium falciparum and Plasmodium vivax. The lack of diagnostic biomarkers for P. vivax, especially those that differentiate the parasite from P. falciparum, significantly hinders P. vivax elimination. Here, we show that P. vivax tryptophan-rich antigen (PvTRAg) can be a diagnostic biomarker for diagnosing P. vivax in malaria patients. We report that polyclonal antibodies against purified PvTRAg protein show interactions with purified PvTRAg and native PvTRAg using Western blots and indirect enzyme-linked immunosorbent assay (ELISA). We also developed an antibody-antigen-based qualitative assay using biolayer interferometry (BLI) to detect vivax infection using plasma samples from patients with different febrile diseases and healthy controls. The polyclonal anti-PvTRAg antibodies were used to capture free native PvTRAg from the patient plasma samples using BLI, providing a new expansion range to make the assay quick, accurate, sensitive, and high-throughput. The data presented in this report provides a proof of concept for PvTRAg, a new antigen, for developing a diagnostic assay for P. vivax identification and differentiation from the rest of the Plasmodium species and, at a later stage, translating the BLI assay into affordable, point-of-care formats to make it more accessible.