In order to establish a method with high-throughput，sensitivity and specificity detecting Brucella，Toxoplasma gondii and Bartonella in pets，specific primers and TaqMan probes were synthesized according to their respective conservative sequences. By optimizing the reaction system，a triple fluorescent PCR assay was established to detect 304 whole blood samples collected from dogs and cats in Xiamen in 2021. The results showed that the method，with good specificity，could specifically amplify the positive nucleic acids of Brucella，Bartonella and Toxoplasma gondii，but failed for other control strains；with strong anti-interference performance，could make different components fail to react with each other when a mixed template was tested；with strong sensitivity，could reach the lowest detection limit of 3×100 copies/μL；with good repeatability，the variable coefficient of intra and inter batch repeated tests were less than 2%. The method could be used to rapidly detect 304 clinical samples within 4 hours，but no positive nucleic acids of Brucella，Toxoplasma gondii and Bartonella were detected. In conclusion，the detection efficiency could be greatly improved by the method，which technically supported early diagnosis and screening of Brucella，Toxoplasma gondii and Bartonella prevalent in various animals.