Toxocariasis is a zoonotic disease caused by Toxocara larvae, transmitted via contaminated environments or infected tissues. Its clinical impact varies among hosts due to differences in larval migration, immune response, and parasite burden. This study aimed to compare the molecular detection efficiency of Toxocara spp. in humans and sheep using the mitochondrial cytochrome c oxidase subunit 1 (COX1) gene as a diagnostic target. A total of 150 blood samples (from humans and sheep with suspected toxocariasis infection) were analyzed. DNA and RNA were extracted and subjected to conventional PCR and quantitative real-time PCR (qPCR) targeting the COX1 gene. Melt curve analysis was conducted to confirmed the product specificity, and the (ΔΔCt) method was used to evaluate relative gene expression. The results demonstrated significantly stronger PCR amplification, clearer bands, and lower Ct values in human samples compared to sheep. qPCR analysis revealed that the COX1 gene expression levels were approximately 99-fold higher in human samples than in sheep, suggesting a higher parasite burden or superior nucleic acid quality. These host-specific differences underline the challenges in detecting Toxocara spp. across different species and emphasize the importance of tailoring diagnostic approaches to each host. Overall, this study highlights the potential use of the COX1 gene as a reliable molecular marker for human toxocariasis diagnosis while noting its reduced sensitivity in sheep. Understanding host-related biological differences is crucial for improving diagnostic accuracy and informing effective parasite control strategies in both human and veterinary medicine.