Abstract The tambaqui C. macropomum is intensively produced in aquaculture and is subject to numerous parasitosis, such as acanthocephalosis, a parasitosis caused by Neoechinorhynchus buttnerae infection. The conventional diagnosis of acanthocephalosis is performed through fish euthanasia, which brings economic loss for fish farmers and poor efficiency in managing disease prevention. Thus, in this study, was proposed the use of molecular tools to develop a nonlethal diagnosis method for acanthocephalosis. For this, a partial gene sequence of the 18S ribosomal RNA gene of N. buttnerae was isolated, and specific primers were designed for the detection of parasite DNA presence in the host's blood by quantitative polymerase chain reaction (qPCR). Infected and uninfected fish were submitted to molecular diagnosis by qPCR, which showed 84% efficiency, 100% specificity and 50% sensitivity. The identification of false negatives led to histopathological analysis. These analyses confirmed the impairment of intestinal structures and the presence of inflammatory response, specific features of acanthocephalosis lesions. Also, gene expression analysis of RAG2 (Recombination Activating Gene) and MALT1 (Mucosa-associated Lymphoid Tissue Lymphoma Translocation) showed a decrease in parasitized fish, demonstrating that the host immune system was compromised. Thus, evidence that it is possible to diagnose non-lethally, by qPCR, the presence of the parasite N. buttnerae from blood samples taken from its host C. macropomum were generated. Experimental alternatives, however, should be improved to increase the sensitivity of the method.