Purpose <p>The aim of the present study was to establish a SYBR Green-based real-time PCR assay for detection of the Nc5 segment from the <i>Neospora caninum</i> genome.</p> Methods <p>The oligonucleotides sequences targeting the Nc5 gene previously reported and designed in-house were validated. Two Primer sets were evaluated and tested in four different combinations. The NP7/NP10 assay was selected and reaction conditions optimized. Efficiency, analytical sensitivity, precision and specificity were assessed. The assay was evaluated in triplicate, in three independent PCR runs performed by two technicians to generate robust results.</p> Results <p>The standard curve determined by tenfold serial dilutions (1 to 1 × 10–⁷) established a reaction efficiency (E) of 102.34%, a correlation coefficient (R²) of 0.999 and a slope of -3.267. LOD of the real-time PCR assay was 0.456 tachyzoites DNA per reaction, as compared to 45.62 for the conventional method. SYBR green real-time PCR was 100 times more sensitive than the conventional method. Precision analysis showed 100% intra- and inter-assay repeatability at the minimum detection limit. The mean assay coefficient of variation (CV%) was 4.19% and standard deviation (SD) 1.67%. No significant differences between the means of <i>Cq</i> in the replicates and technicians (P &gt; 0.05) was found, indicating that the assay is robust and accurate. The applicability of the assay was tested and <i>N. caninum</i> DNA was detected in milk, blood, amniotic fluid, placenta and different tissue samples.</p> Conclusion <p>The protocol had high specificity, confirmed by melting curve analysis and no cross-reactions with other tested microorganisms. The SYBR Green-based PCR protocol standardized in this study is a highly sensitive and specific method, reproducible and applicable for the detection of <i>N. caninum</i> in different biological samples.</p>