Tick-borne pathogens, particularly Babesia and Theileria species, are major threats to cattle production, causing economically significant diseases such as babesiosis and theileriosis. In this study, a real-time SYBR Green PCR assay was developed to detect Babesia and Theileria species in hard ticks (N = 65) and cattle blood samples (N = 143) from Thailand. Using primers targeting the mitochondrial cytochrome b gene for Babesia and the nuclear 18S rRNA gene for Theileria, the assay measured specific melting temperatures (Tm) for each species. The results showed distinct Tm values for Babesia bigemina (74.38 ± 0.04 °C), Babesia bovis (75.7 ± 0.06 °C), Theileria orientalis (74.61 ± 0.03 °C), Theileria sinensis (75.84 ± 0.03 °C), and Theileria annulata (74.06 ± 0.03 °C). The assay demonstrated high specificity, with a cutoff cycle threshold of < 35 cycles and a minimum detectable concentration of 10 copies/μL. Significant species differences in melting curves were confirmed using Tukey's HSD test (p < 0.05). Theileria orientalis was detected in 8.4% of cattle blood samples, while T. sinensis was found in 25.9%, and B. bigemina in 0.7%. Theileria orientalis was also detected in 7.7% of tick samples, T. sinensis in 16.9%, and B. bigemina in 6.1%. The assay returned negative results for all non-target blood and tissue pathogens tested for specificity. This robust, high-throughput assay is highly effective for monitoring Babesia and Theileria infections, facilitating close surveillance and intervention efforts against tick-borne diseases in cattle.